Characterization of a P-type Na 1 -ATPase of a Facultatively Anaerobic Alkaliphile, Exiguobacterium aurantiacum *

A facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum , possesses a P-type Na 1 -stimulated ATPase in the membrane (Koyama, N. (1999) Curr. Mi-crobiol. 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 m M ) and forms an acylphosphate intermediate, suggesting a P-type ATPase. The enzyme, when reconstituted into soybean phospholipid vesicles, exhibited ATP-depend-ent 22 Na 1 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 1 .

Purification of the ATPase-The freshly prepared membranes (about 60 mg of protein) were treated with 10 mM EDTA to remove loosely bound membrane proteins. After treatment, the membranes were suspended in 20 ml of a buffer containing 20 mM Tris-HCl (pH 8), 200 mM KCl, and 1 mM MgCl 2 , and then 2.9 ml of 20 mM deoxy-BIGCHAP was added under magnetic stirring at 4°C. To the supernatant obtained by the centrifugation for 20 min at 90,000 ϫ g, ammonium sulfate was added to 85% saturation. The floating precipitate was collected by centrifugation and then resolved in 0.5 ml of H 2 O. The sample thus 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 MgCl 2 , 1 mM deoxy-BIGCHAP, and soybean lecithin (0.5 mg/ml), and then fractions of 1 ml were collected. Active fractions (14 -17) were combined, and 500 mM potassium phosphate (pH 7.5) was added to give a final concentration of 25 mM. The solution was applied to a column (2 ϫ 6 cm) of hydroxyapatite (Gigapite Biochemicals, Tokyo, Japan), which was equilibrated with a buffer containing 25 mM potassium phosphate (pH 7.5) and 1 mM deoxy-BIGCHAP. The column was washed with 5 ml of the same buffer, and the enzyme was eluted with a 30-ml linear gradient of 25 mM potassium phosphate (pH 7.5) and 700 mM potassium phosphate (pH 8.5). About 30 fractions of 1 ml were collected, and to each of them, 0.5 mg of soybean lecithin was immediately added. The most active fractions (24 -28) were combined and dialyzed against 20 mM Tris-HCl (pH 8.5), 200 mM KCl, and 1.0 mM MgCl 2 (Buffer A) for the characterization of the enzyme.
ATPase Assay-The ATPase activity was assayed by the two different procedures. Routinely, the release of inorganic phosphate by the hydrolysis of ATP at 30°C was measured as described previously (22). The reaction was carried out in a final volume of 1 ml of 20 mM Tris-HCl buffer (pH 8.5) containing 2 mM ATP, 3 mM MgCl 2 and 200 mM NaCl unless otherwise stated. One enzyme unit was defined as 1 mol of ATP hydrolyzed/min.
When the activity of the fractions from hydroxyapatite chromatography was measured, the coupled enzyme assay was employed. The assay was carried out by continuously monitoring the oxidation of NADH at 340 nm with a linked enzyme system as described previously (23). The reaction medium contained 20 mM Tris-HCl (pH 8.5), 200 mM NaCl, 2 mM ATP, 3 mM * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Phosphorylation Reaction-Phosphorylation was conducted as described previously (20). Briefly, the purified enzyme (about 1 g of protein) was incubated at 0°C in a total volume of 50 l of 20 mM Tris-HCl (pH 8.2), 100 mM KCl, 100 mM NaCl, 1 mM MgCl 2 , and 1 mM ␤-mercaptoethanol. The reaction was initiated by the addition of 10 M [␥-32 P]ATP (111 TBq/mmol). After 60 s, the reaction was stopped by adding 50 l of 10% trichloroacetic acid containing 1 mM sodium phosphate. The precipitated material was washed with water and then subjected to acidic lithium dodecyl sulfate-polyacrylamide gel electrophoresis (25) followed by autoradiography. Some precipitated materials were subjected to 0.1 M Na 2 CO 3 at 0°C for 5 min and to 0.25 M hydroxylamine at 25°C and pH 5.2 for 20 min, respectively, followed by trichloroacetic acid precipitation. Further treatment was the same as for the untreated samples.
Reconstitution of the Purified ATPase-Reconstitution was carried out as described previously (20). Briefly, to 200 l of Buffer A containing 1.6 mg of soybean lecithin, which was dissolved by sonication from a dried film deposited in the tube from an ether solution, was added 67 l of 20 mM CHAPS. The suspension was mixed with 1 ml of the purified enzyme (about 6 g), and then 160 mg of SM-2 Bio-Beads (Bio-Rad), which was previously washed by the procedure of Holloway (26), was added. After 30 -60 min of a continuous stirring at room temperature, the turbid sample was applied instantly for the assay.
Measurements of 22 Na ϩ Transport and Membrane Potential of the Reconstituted Vesicles-The assay of 22 Na ϩ transport was conducted by the same procedure described previously (20).
Membrane potential was measured by using Oxonol V at excitation and emission wavelengths of 595 and 640 nm, respectively, in a final volume of 2 ml of Buffer A containing 500 l of the reconstituted sample.
Protein Determination-Protein concentration was determined by the Lowry assay (27) using bovine serum albumin as standard.

RESULTS AND DISCUSSION
For the recovery of Na ϩ -stimulated ATPase activity in a soluble fraction from the membranes, it was necessary to use detergent, suggesting that the enzyme is an integral membrane protein. Several detergents, such as Triton X-100, heptyl thioglucoside, polyoxyethylene 9-lauryl ether (C 12 E 9 ), decanoyl N-methylglucamide, n-nonyl-␤-D-thiomaltoside, sucrose monocaprate, sodium cholate, CHAPS, BIGCHAP, and deoxy-BIG-CHAP were tested for the solubilization. Among them, deoxy-BIGCHAP and CHAPS were most effective. Because CHAPS, an ionic detergent, is inadequate to the application to ionexchange chromatography for purification, a nonionic deoxy-BIGCHAP was used for solubilization. Fig. 1A depicts the results of SDS-polyacrylamide gel electrophoresis at different stages of the purification procedure. A 100-kDa polypeptide appeared as a major band, although there are still some faint bands, the number of which varied from one preparation to the next. Incubation of the purified sample with [␥-32 P]ATP demonstrated the existence of phosphorylated intermediate with a molecular mass of 100 kDa by acidic gel electrophoresis (Fig. 1B, lane 1). Treatment of the labeled samples with 0.1 M Na 2 CO 3 and 0.25 M hydroxylamine, respectively, released the radioactive phosphates (lanes 2 and 3), 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.
The purified enzyme utilized ATP as the best substrate among the nucleotides tested. The rates of hydrolysis of CTP, ITP, dTTP, UTP, ADP and AMP relative to ATP were 8, 5, 5, 4, 0, and 0%, respectively. The enzyme is likely to hydrolyze only triphosphates of nucleosides. Fig. 2 depicts the effect of pH on the ATPase activity. The enzyme exhibited an optimum pH for activity at around 9. Fig. 3 depicts the concentration effect of vanadate on the ATPase activity. When vanadate concentration in the reaction medium was increased, ATPase activity was decreased sharply in the concentration range of 0.1-10 M, and a half-maximal inhibition occurred at 3 M, which was slightly higher than that of the membrane-bound ATPase (20).
The ATPase activity of the purified enzyme depended on NaCl concentration (Fig. 4). When measured in the presence of various concentration of NaCl, the activity was increased with increasing NaCl concentration, with near-saturation at around 100 mM.
The reconstituted vesicles of enzyme exhibited a significant uptake of 22 Na ϩ when ATP was added, which was completely inhibited by gramicidin (Fig. 5). The ATP-dependent uptake of Na ϩ is likely to be accompanied with generation of an interior positive membrane potential, which was suggested by quenching of Oxonol V, a fluorescent dye, when NaCl was added (data not shown). Similarly to the ATPase activity, the initial rate of quenching was increased with increasing NaCl concentration (Fig. 4). The membrane potential could be dissipated by gramicidin, whereas the ATPase activity was accelerated about 3-fold by 10 Ϫ7 M gramicidin. Addition of LiCl, KCl, and RbCl caused essentially no quenching, suggesting that the quenching by NaCl is specific effect of Na ϩ . These facts may indicate an electrogenic transport of Na ϩ by the enzyme.
The result obtained in this study revealed that E. aurantiacum BL77/1 possesses a P-type Na ϩ -transport ATPase. The growth medium of the bacterium contained approximately 0.5 M Na ϩ . When Na ϩ in the medium was replaced by K ϩ , essentially no growth was observed. The bacterium exhibited Na ϩdependent uptake of amino acids such as leucine and serine, 2 which suggests that ⌬ Na ϩ is utilized as a driving force of active transport of the amino acids. The purified ATPase may contribute to the generation of ⌬ Na ϩ. Na ϩ /K ϩ -ATPase, a sodium pump in the plasma membrane of animal cells, is a typical P-type ATPase. In a preliminary experiment, the purified enzyme did not cross-react with an antiserum against porcine Na ϩ /K ϩ -ATPase. The enzyme might possess no common epitope with animal sodium pump. We are now attempting to isolate the structural gene of the enzyme for further characterization.