Abstract
A facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, possesses a P-type Na(+)-stimulated ATPase in the membrane (Koyama, N. (1999) Curr. Microbiol. 39, 27-30). In this study, we attempted to purify and characterize the enzyme. The ATPase appears to consist of a single polypeptide with an apparent molecular mass of 100 kDa. The enzyme exhibited an optimum pH for activity at around 9. The enzyme was strongly inhibited by vanadate (50% inhibition observed at 3 microm) and forms an acylphosphate intermediate, suggesting a P-type ATPase. The enzyme, when reconstituted into soybean phospholipid vesicles, exhibited ATP-dependent (22)Na(+) uptake, which was completely inhibited by gramicidin. The reconstituted vesicles exhibited a generation of membrane potential (positive, inside). The enzyme is likely to be involved in an electrogenic transport of Na(+).
Highlights
It has been suggested that a facultatively anaerobic alkaliphile BL77/1, a strain of Exiguobacterium aurantiacum, possesses a Naϩ-stimulated P-type ATPase in the membrane, which is expected to function as a sodium pump [20]
The enzyme appears to consist of a single polypeptide with an apparent molecular mass of 100 kDa
Treatment of the labeled samples with 0.1 M Na2CO3 and 0.25 M hydroxylamine, respectively, released the radioactive phosphates, indicating the acylphophate intermediate [28]. These results suggest that the purified enzyme is a P-type ATPase that consists of a single polypeptide with an apparent molecular mass of 100 kDa
Summary
It has been suggested that a facultatively anaerobic alkaliphile BL77/1, a strain of Exiguobacterium aurantiacum, possesses a Naϩ-stimulated P-type ATPase in the membrane, which is expected to function as a sodium pump [20]. The membranes were suspended in 20 ml of a buffer containing 20 mM Tris-HCl (pH 8), 200 mM KCl, and 1 mM MgCl2, and 2.9 ml of 20 mM deoxy-BIGCHAP was added under magnetic stirring at 4 °C. The sample obtained was applied on a Sepharose S-300 column (2 ϫ 20 cm), which was equilibrated with a buffer (pH 8) containing 20 mM Tris, 20% ammonium sulfate, 1 mM MgCl2, 1 mM deoxyBIGCHAP, and soybean lecithin (0.5 mg/ml), and fractions of 1 ml were collected.
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