Presence of a Na⁺-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacteriumAphanothece halophytica

Abstract

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.

Molecular biology of cyanobacterial salt acclimation

“…The direct salt-stimulated extrusion of protons was attributed to an increased respiratory activity or stimulated activities of Ptype H 1 -ATPases (Nitschmann & Peschek, 1985;Molitor et al, 1986;Gabbay-Azaria et al, 1992Jeanjean et al, 1993;Wiangnon et al, 2007). It is believed that Na 1 /H 1 antiporters use the proton motive force or the membrane potential at the cytoplasmic membrane for the uphill export of Na 1 in saline medium.…”

Na+/H+ exchange activity in the alkaliphile halotolerant cyanobacterium Aphanothece halophytica

“…Moreover, overexpression of NhaP conferred salt tolerance of the fresh-water cyanobacterium Synechococcus rendering its growth even in sea water (Waditee et al 2002). Recently, we have shown the existence of a H + -ATPase specifically stimulated by Na + in A. halophytica and that this H + -ATPase could provide the driving force for Na + transport (Wiangnon et al 2007). In the marine cyanobacterium, Spirulina subsalsa, proton gradient generation via cytochrome oxidase has been suggested to play a role in Na + extrusion by means of Na + /H + exchange (GabbayAzaria et al 1992).…”

Na+-stimulated ATPase of alkaliphilic halotolerant cyanobacterium Aphanothece halophytica translocates Na+ into proteoliposomes via Na+ uniport mechanism

“…Intracellular cation levels for A. halophytica mainly Na + and K + have been reported to be within the range of 260-420 mM depending on the external salinities [26]. Previously we reported the presence of a Na + -stimulated ATPase in the plasma membrane of A. halophytica [27]. The membrane vesicles could take up Na + when supplied with ATP.…”