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Characterization of NA⁺-ATPase and its role in salinity tolerance of a halotolerant cyanobacterium Aphanohece halophytica |
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| รหัสดีโอไอ | |
| Title | Characterization of NA⁺-ATPase and its role in salinity tolerance of a halotolerant cyanobacterium Aphanohece halophytica |
| Creator | Kanteera Soontharapirakkul |
| Contributor | Aran Incharoensakdi, Teruhiro Takabe |
| Publisher | Chulalongkorn University |
| Publication Year | 2553 |
| Keyword | Sodium/potassium ATPase -- Properties, Cyanobacteria, Salinity, โซเดียม/โพแทสเซียมเอทีพีเอส -- คุณสมบัติ, ไซยาโนแบคทีเรีย, ความเค็ม |
| Abstract | ATPase was purified from Aphanothece halophytica membrane vesicles with a 17.5–fold purification with 6.5% yield. The purified enzyme catalyzed the hydrolysis of ATP in the presence of Na+. The apparent K[subscript m] values for Na⁺ and ATP were 2.0 and 1.2 mM, respectively. The enzyme is likely the F-type ATPase based on the usual subunit pattern from SDS-PAGE and inhibitors study. The purified enzyme reconstituted into liposomes functions as an electrogenic Na⁺ pump which transports Na⁺ upon hydrolysis of ATP and then a secondary event, Na+- and ATP-dependent H⁺ efflux from proteoliposomes, is driven by the electric potential generated by Na⁺-stimulated ATPase.A putative F-type Na⁺-atp operon coding for Na+-ATP synthase (Na⁺-ATPase) from A. halophytica was isolated. The operon consists of nine genes which encode putative subunits , , I, hypothetical protein, a, c, b, , and . The expression plasmid (pTrcHis2C-ApNa⁺-atp) was constructed and transformed into E. coli mutant DK8 (atp) deficient in ATP synthase. The inverted membrane vesicles from ApNa⁺-ATPase-expressing E. coli DK8 cells exhibited Na⁺-dependent ATP hydrolysis activity, which was inhibited by the sodium gradient dissipator monensin and the F-type ATPase inhibitor tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na⁺ ion protected the inhibition of ApNa⁺-ATPase by N,N'-dicyclohexylcarbodiimide (DCCD). The ATP synthesis activity was also observed using the Na⁺-loaded inverted membrane vesicles from ApNa⁺-ATPase-expressing E. coli DK8 cells. Expression of ApNa+-atp operon in a heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional F-type Na⁺-dependent ATPase playing a potential role of salt-stress tolerance. |
| URL Website | cuir.car.chula.ac.th |
