There have been studies of ATPases associated with various cations such as Ca2 , Cu2 , K and Mg2 in a number of cyanobacteria . However, ATPase involved in Na transport has not been directly demonstrated experimentally in cyanobacteria. In this study, we focused on the involvement of ATPase in Na homeostasis in the alkaliphilic halotolerant cyanobacterium A. halophytica. High ATPase activity detected in membrane vesicles of cells grown under high salinity and high pH conditions suggested that ATPase from A. halophytica plays a role in the response of cells against an increase in salinity and pH. Similar observations were found in Streptococcus faecalis, Tetraselmis viridis, and Heterosigma akashiwo . Furthermore, we also found that A. halophytica requires Na for its growth. The presence of gramicidin D, an ionophore that dissipates Na gradients, in the growth medium resulted in the cessation of cell growth .
These results are in agreement with those previously reported in a facultative anaerobic alkaliphile M 12 which utilized sodium motive force generated by a sodium pump for active transport of solutes while other alkaliphiles produced B-Raf inhibitor Na by a Na H antiporter which is a secondary transport system for Na . To investigate Na transport in relation to ATPase activity in A. halophytica, the ATPase was purified and was tentatively classified as an F type ATPase. This is based on the results that its activity was inhibited by azide and DCCD, which are inhibitors of F1F0 ATPase but not by orthovanadate and nitrate which are inhibitors of P type and V type ATPases, respectively . DCCD is believed to inhibit F type ATPase due to the binding of DCCD to a highly conserved carboxyl residue in the c subunit of the F0 portion . The band pattern of the purified enzyme analyzed by SDSPAGE as shown in Figure 2 was comparable to the typical mobilities and band pattern of F type ATPase subunits from both Ilyobacter tartaricus and a thermoalkaliphilic Bacillus sp. strain TA2.A1 .
The polypeptide bands could tentatively be identified as ATPase subunits of an F type ATPase, namely: a , b , g , a , b ? and c . Moreover, the band tentatively identified as F type ATPase subunit c of A. halophytica was confirmed by LC MS MS analysis. One of the obtained sequences after trypsin digestion namely ISSGAEGIAR kinase inhibitor selleck was found to be highly identical to the partial sequence of F type ATPase subunit c of at least four strains of cyanobacteria, namely Synechococcus sp. WH 8102, Synechococcus sp. CC9902, Synechococcus sp. PCC 7002 and Gloeobacter violaceus. Overall, the results from the inhibitor effects, and the typical subunits band pattern as well as the protection by Na against DCCD inhibition of ATPase activity , suggest that the ATPase from A. halophytica is likely a member of the F type ATPases.