Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin

Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellular homeostasis. We have previously identified in human tissues a transporter, DNP-SG [S-(2, 4-dinitrophenyl)glutathione] ATPase, capable of carrying out this function [Awasthi et al. (1998) Biochemistry 37, 5231-5238, 5239-5248]. We now report the cloning of DNP-SG ATPase. The sequence of the cDNA clone was identical to that of human RLIP76, a known Ral-binding protein. RLIP76 expressed in E. coli was purified by DNP-SG affinity chromatography. Purified recombinant RLIP76: (1) had ATPase activity stimulated by DNP-SG or doxorubicin (DOX), and the K(m) values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well as with defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameters similar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistance to DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesicles from transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donor proteoliposomes confers DOX resistance. These results indicate that RLIP76, in addition to its role in signal transduction, can catalyze transport of glutathione conjugates and xenobiotics, and may contribute to the multidrug resistance phenomenon.     

RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes.

We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al. Biochemistry 39, 9327-9334). Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes. In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes. Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function. Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70%. On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes.     

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76)

We have recently demonstrated that a previously known Ral-binding GTPase activating protein, RLIP76, can also catalyze ATP-dependent transport of various structurally unrelated xeno- and endobiotics irrespective of their net charge (Awasthi et al., 2000, Biochemistry, 39: 9327). RLIP76 is a non-ATP binding cassette (ABC) protein but it has two ATP-binding sites and shows basal ATPase activity which is stimulated in the presence of its transport substrates (allocrites) such as doxorubicin (DOX) and S-(2,4-dinitrophenyl) glutathione (DNP-SG). Proteoliposomes reconstituted with purified RLIP76 catalyze ATP-dependent, saturable transport of DOX, as well as of glutathione-conjugates including leukotrienes (LTC4) and the GSH-conjugate of 4-hydroxynonenal (GS-HNE). In erythrocytes the majority of transport activity for DOX, GS-HNE, and LTC4 is accounted for by RLIP76. Cells exposed to mild oxidative stress show a rapid and transient induction of RLIP76 resulting in an increased efflux of GS-HNE and acquire resistance to oxidative stress mediated toxicity and apoptosis. Cells transfected with RLIP76 acquire resistance to DOX through increased efflux of the drug suggesting its possible role in the mechanisms of drug-resistance. In this article, we discuss the significance of transport functions of RLIP76 highlighting its role in the defense mechanisms against oxidative injury, and modulation of signaling mechanisms.     

Functional reconstitution of Ral-binding GTPase activating protein,RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal

Earlier studies from our laboratories have shown that RLIP76, a previously described Ral-binding GTPase activating protein (Jullien-Flores et al., 1995, J. Biol. Chem. 270: 22473), is identical with the xenobiotic transporter DNP-SG ATPase, and can catalyze ATP-dependent transport of glutathione-conjugates as well as doxorubin (Awasthi et al., 2000, Biochemistry, 39: 9327). We have now reconstituted purified bacterially expressed RLIP76 in proteoliposomes, and have studied ATP-dependent uptake of the glutathione conjugate of 4-hydroxynonenal (GS-HNE) by these vesicles. Results of these studies show that RLIP76 reconstituted in proteoliposomes catalyzes ATP-dependent transport of GS-HNE against a concentration gradient. The transport of GS-HNE is saturable with respect to ATP as well as GS-HNE with K(m) values of 1.4mM and 2.5 microM, respectively. These studies demonstrate that RLIP76 mediates active transport of GS-HNE, and are consistent with our previous work showing that RLIP76-mediated efflux of GS-HNE regulates the intracellular concentration of 4-HNE and thereby affects 4-HNE mediated signaling.     

Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences

We have recently shown that RLIP76, a Ral-binding, GTPase-activating protein, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathione conjugates [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334]. RLIP76 overexpressed in human cells or transformed E. coli undergoes proteolysis to yield several fragments, including two prominent peptides, N-RLIP76(1-367) and C-RLIP76(410-655), from the N- and C-terminal domains, respectively. To investigate whether the fragmentation of RLIP76 has any relevance to its transport function, we have studied the characteristics of these two peptide fragments. Recombinant N-RLIP76(1-367) and C-RLIP76(410-655) were purified from overexpressing transformed E. coli. While N-RLIP76(1-367) readily underwent proteolysis, showing SDS-gel patterns similar to those of RLIP76, C-RLIP76(410-655) was resistant to such degradation. Both N-RLIP76(1-367) and C-RLIP76(410-655) had ATPase activity (K(m) for ATP, 2.5 and 2.0 mM, respectively) which was stimulated by DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinity labeling with 8-azido-ATP that was increased in the presence of compounds that stimulated their ATPase activity. Photoaffinity labeling was also increased in the presence of vanadate, indicating trapping of a reaction intermediate in the ATP binding site. The ATP binding sites in N-RLIP76(1-367) and C-RLIP76(410-655) were identified to be (69)GKKKGK(74) and (418)GGIKDLSK(425), respectively. Mutation of K(74) and K(425) to M residues, in N-RLIP76(1-367) and C-RLIP76(410-655), respectively, abrogated their ATPase activity as well as azido-ATP labeling. Proteoliposomes reconstituted with either N-RLIP76(1-367) or C-RLIP76(410-655) alone did not catalyze ATP-dependent transport of DOX or COL. However, proteoliposomes reconstituted with a mixture of N-RLIP76(1-367) and C-RLIP76(410-655) mediated such transport. Proteoliposomes reconstituted with the mixture of mutant peptides lacking ATPase activity did not exhibit transport activity. Present studies have identified the ATP binding sites in RLIP76, and show that DOX and COL transport can be reconstituted by two fragments of RLIP76.     

POB1 over-expression inhibits RLIP76-mediated transport of glutathione-conjugates, drugs and promotes apoptosis

RLIP76 (RALBP1) is a Ral-binding nucleotidase which functions as an energy-dependent transporter for glutathione (GSH)-conjugates as well as structurally unrelated xenobiotics. Partner of RALBP1 (POB1), also referred to as REPS2, was identified as the human RLIP76-binding protein, which contains a coiled-coil C-terminal region that binds with the RLIP76. Recent studies show that over-expression of POB1 in prostate cancer cells induces apoptosis. In present studies, we have purified POB1 and one of its deletion mutants POB1(1-512) (lacking the RLIP76-binding domain), and examined their effect on the transport activity of RLIP76. Both doxorubicin and a model GSH-conjugate, dinitrophenyl-S-glutathione (DNP-SG), transport were inhibited by POB1 in a concentration-dependent manner but not by POB1(1-512), lacking RLIP76-binding site. Liposomal delivery of recombinant POB1 to H358 (NSCLC) cancer cells caused apoptosis in a concentration-dependent manner, whereas the POB1 mutant deficient in RLIP76-binding site did not exert this effect. Augmentation of cellular POB1 resulted in increased intracellular DOX-accumulation as well as decreased rate of efflux from cells. These results show for the first time that POB1 can regulate the transport function of RLIP76 and are consistent with our previous studies showing that inhibition of RLIP76 induces apoptosis in cancer cells through the accumulation of endogenously formed GSH-conjugates.     

Diminished drug transport and augmented radiation sensitivity caused by loss of RLIP76

This study was undertaken to characterize the consequences of Ral-interacting protein (RLIP76)-loss with respect to drug resistance, transport, radiation resistance, and alternative transport mechanisms in mouse embryonic fibroblasts (MEFs). MEFs were derived from RLIP76+/+, RLIP76+/- and RLIP76-/- mice. The transport of doxorubicin (DOX), colchicine (COL), leukotriene C4 and dinitrophenyl S-glutathione (DNP-SG) was analyzed in inside-out vesicles (IOVs) prepared from MEFs. We used immuno-titration of transport activity to determine the contribution of RLIP76, MRP1, and p-glycoprotein (Pgp) towards total transport activity. Loss of RLIP76 alleles resulted in significant sensitization to radiation, DOX, cisplatin, and vinorelbine (VRL). In IOVs prepared from MEFs, we observed a stepwise loss of transport activity. Loss of RLIP76 confers sensitivity to xenobiotics and radiation due to the loss of a common transport mechanism for glutathione-electrophile conjugates and xenobiotics.     

Purification and reconstitution of functional human P-glycoprotein

The overexpression of the P-glycoprotein, theMDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [³H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.     

Identification of P-57, a serine proteinase, from human erythrocyte membranes, which cleaves both chains of human third component (C3) of complement

A proteinase, which cleaves human third component of complement, was solubilized from erythrocyte membranes then purified by gel filtration chromatography, fluid phase electrophoresis, and hydroxylapatite chromatography. Labeling of the purified material by 125I or 3H-DFP and measurement of proteolytic activity subsequently isolated by SDS-polyacrylamide gel electrophoresis allowed to identify a 57 kDa single band, in non reducing conditions. Inhibition of this activity by PMSF supports covalent modification of an active serine residue. This membrane serine proteinase cleaved alpha and beta chains of human third component of complement, suggesting that p-57 is distinct from plasma serine proteinases.     

Gender related differences in ATP-dependent transport of dinitrophenyl-glutathione conjugate across murine canalicular liver plasma membrane

The present study reports gender related differences in ATP-dependent transport of dinitrophenyl-glutathione (GSH) conjugate (DNP-SG), a model GSH xenobiotic conjugate, across murine canalicular liver plasma membrane (cLPM). ATP-dependent transport of DNP-SG across female A/J mouse cLPM was mediated by two components, a high-affinity and a low-affinity component, with corresponding Km of 18 microM (Vmax 0.02 nmol/min.mg) and 500 microM (Vmax 0.23 nmol/min.mg), respectively. On the other hand, only one component for the ATP-dependent transport of DNP-SG was observed in male mouse cLPM (K(m) 130 microM; Vmax 0.18 nmol/min.mg). Moreover, the rate of ATP-dependent transport of DNP-SG was markedly higher in the cLPM fraction of male mouse compared with that of the female. Presence of two transport components in female mouse cLPM, but only one system in the cLPM fraction of male mouse, was confirmed by measuring DNP-SG mediated stimulation of ATP hydrolysis (DNP-SG ATPase activity). To the best of our knowledge, the present study is the first report on gender related differences in ATP-dependent murine canalicular transport of GSH conjugates.     

The role of PKCalpha and RLIP76 in transport-mediated doxorubicin-resistance in lung cancer

In deletion mutant analyses of potential phosphorylation sites in RLIP76, we identified T297 and S509 as targets for phosphorylation by PKCalpha. Phosphorylation at T297 increased doxorubicin (DOX)-transport activity approximately 2-fold for RLIP76 purified from recombinant source, or from three small (H69, H1417, H1618) and three non-small cell, one each derived from H226 (squamous), H358 (bronchio alveolar), and H1395 (adenocarcinoma) lung cancer cell lines. T297 phosphorylation conferred sensitivity to tryptic digestion at R293. The specific activity for DOX-transport by RLIP76 purified from non-small cell, which was primarily in the phosphorylated form, was approximately twice that in small cell lung cancer cell lines. These finding offer a novel explanation for the observed intrinsic differences in sensitivity to DOX between non-small cell and small cell lung cancer cell lines.     

Determinants of differential doxorubicin sensitivity between SCLC and NSCLC

Doxorubicin (DOX) transport activity of Ral-interacting protein (RLIP76) in non-small cell lung cancer (NSCLC) is approximately twice that of in small cell lung cancer (SCLC). Since protein-kinase-C (PKC)alpha mediated phosphorylation of RLIP76 causes doubling of the specific activity of RLIP76, and NSCLC cells are known to have greater PKCalpha activity, we examined the contribution of PKC mediated phosphorylation of RLIP76 towards intrinsic DOX-resistance in human NSCLC. Expression of a deletion mutant RLIP76(delPKCalpha-sites) followed by depletion of the wild-type RLIP76 using a siRNA targeted at one of the deleted regions resulted in generation of cells expressing only the mutant protein, which could not be phosphorylated by PKCalpha. DOX-transport activity of the mutant RLIP76 purified from NSCLC and SCLC was similar and comparable to that of RLIP76 purified from the wild-type SCLC. However, this activity was significantly lower than that of RLIP76 purified from the wild-type NSCLC. After siRNA mediated depletion of PKCalpha, DOX-transport activities of RLIP76 purified from SCLC and NSCLC were indistinguishable. Depletion of PKCalpha inhibited the growth of NSCLC more than SCLC cells (70+/-3% vs. 43+/-5%, respectively). PKCalpha-depletion lowered the IC(50) of NSCLC cell lines for DOX to the same level as that observed for SCLC. RLIP76(-/-) mouse embryonic fibroblasts (MEFs) were significantly more sensitive to DOX as compared with RLIP76(+/+) MEFs (IC(50) 25 vs. 125nM, respectively). However, PKCalpha-depletion did not affect DOX-cytotoxicity towards RLIP76(-/-) MEFs, as opposed to RLIP76(+/+) MEFs which were sensitized by 2.2-fold. These results demonstrate that RLIP76 is a primary determinant of DOX-resistance, and that PKCalpha mediated accumulation defect and DOX-resistance in NSCLC is primarily due to differential phosphorylation of RLIP76 in SCLC and NSCLC.     

Nucleotide-induced conformational changes in the human multidrug resistance protein MRP1 are related to the capacity of chemotherapeutic drugs to accumulate or not in resistant cells

Intracellular accumulation of anthracycline derivatives was measured in a human embryonic kidney cell line (HEK) and a resistant subline (HEK/multidrug resistance protein (MRP1)) overexpressing MRP1 at the plasma membrane surface. Two compounds (daunorubicin and doxorubicin) were rejected outside the multidrug-resistant cells. On the contrary, three compounds (4'-deoxy-4'-iodo-doxorubicin, 4-demethoxy-daunorubicin and 3'-(3-methoxymorpholino)doxorubicin) accumulated equally within sensitive HEK cells and resistant HEK/MRP1 cells. Our main objective here was to characterize the MRP1 conformational changes mediated by the binding of these anthracycline derivatives and to determine whether these conformational changes are related to MRP1-mediated drug transport. MRP1 was reconstituted in lipid vesicles as previously described [Manciu, L., Chang, X.B., Riordan, J.R. and Ruysschaert, J.-M. (2000) Biochemistry 39, 13026-13033]. The reconstituted protein was shown to conserve its ATPase and drug transport activity. Acrylamide quenching of Trp fluorescence was used to monitor drug-dependent conformational changes. Binding of drugs (4-demethoxy-daunorubicin and 3'-(3-methoxymorpholino)doxorubicin) which accumulate in resistant cells immobilizes MRP1 in a conformational state that is insensitive to ATP binding whereas drugs rejected outside the resistant cells (daunorubicin, doxorubicin) favor a conformational change which may be a required step in the transport process.     

Membrane ATPase of Vibrio alginolyticus. Ion transport activity and homology with F0F1-ATPase from E. coli]

F0F1-ATPase has been isolated from the marine alkali-resistant bacterium Vibrio alginolyticus. The enzyme subunits cross-reacted with antibodies against subunits alpha, beta, gamma, epsilon, and b of E. coli ATPase. The purified ATPase was reconstituted into liposomes effecting an ATP-dependent uptake of H+. Proton transport was inhibited by the ATPase blockers DCCD, triphenyltin, and venturicidin. Na+ ions had no effect on ATP-dependent proton transport. No ATP-dependent transport of Na+ was detected in proteoliposomes.     

Mitogenic and drug-resistance mediating effects of PKCalpha require RLIP76

PKCalpha-activation is a key signaling event governing cell growth, stress-resistance, and drug-resistance. Our recent studies demonstrated that DOX-resistance mediating effects of PKCalpha require the presence of RLIP76, and their concerted action is sufficient to explain intrinsic DOX-resistance of NSCLC [S.S. Singhal, D. Wickramarachchi, J. Singhal, S. Yadav, Y.C. Awasthi, et al., Determinants of differential doxorubicin sensitivity between SCLC and NSCLC. FEBS Lett. 580 (2006) 2258-2264]. Present studies were carried out to further explore the suggestion from the previous studies that the mitogenic effects of PKCalpha also require RLIP76. RLIP76-/- MEFs were resistant to PKCalpha-depletion mediated growth inhibition, as well as to the PKCalpha-dependent mitogen, phorbol 12-myristate 13-acetate (PMA). Augmenting cellular levels of RLIP76 using purified recombinant RLIP76 increased growth rate in all cells, and restored the sensitivity of RLIP76-/- MEFs to both inhibition through PKCalpha-depletion and stimulation through PMA. These results show that RLIP76 is a necessary down-stream effector for PKCalpha-mediated mitogenesis.     

Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.     

Some properties of membrane-bound, solubilized and reconstituted into liposomes H+-ATPase of vacuoles of Saccharomyces carlsbergensis

Vacuoles of yeast grown in peptone medium possessed high ATPase activity (up to 1 mumol X mg protein-1 X min-1). Membrane-bound and solubilized ATPase activities were insensitive to vanadate and azide, but were inhibited by NO-3 . K+ and cyclic AMP stimulated both membrane-bound and solubilized ATPase activities. Dio-9 activated the membrane form of vacuolar ATPase 1.5-2-fold and did not affect the solubilized enzyme. Solubilized and partially purified vacuolar ATPase was reconstituted with soy-bean phospholipids by a freeze-thaw procedure. ATPase activities in native vacuoles and proteoliposomes were stimulated effectively by Dio-9, the protonophore FCCP and ionophores valinomycin and nigericin. ATP-dependent H+ transport into proteoliposomes was also shown by quenching of ACMA fluorescence. Vacuolar and partially purified ATPase preparations possessed also GTPase activity. Unlike ATPase, however, GTPase was not incorporated as a proton pump into liposomes.     

Reconstitution of an ATP-dependent sodium pump with an ATPase from electric eel and pure phospholipids.

An ATP-dependent sodium pump was reconstituted with an ATPase preparation from the electric eel and purified phospholipids. Vesicles reconstituted by the sonication procedure catalyzed Na⁺ translocation at a rate 10 to 20 times higher than vesicles reconstituted by the cholate-dialysis procedure. A small but reproducible stimulation of Na⁺ transport by valinomycin in the presence of K⁺ was observed.     

Characterization of reconstituted plasma membrane H+-ATPase from maize roots

Corn ( Zea mays L.) plasma membranes from KI-washed microsomal fractions were further purified by isopycnic sucrose density centrifugation. An examination of separated fractions indicated that vesicles with nitrate-insensitive proton transport copurified with fractions containing vanadate-sensitive ATPase activity. The ATPase in purified plasma membrane was reconstituted into liposomes by a detergent dilution technique using deoxycholate. The reconstituted ATPase exhibited characteristics similar to those of the native enzyme. However, reconstituted preparations showed an enhanced sensitivity to vanadate, a diminished phosphatase activity and a high specific rate of ATP-dependent H⁺-transport. Apparent K_i values of reconstituted and native enzymes with respect to vanadate were 20 and 50 μ M , respectively; the KJ value of the H+-pumping of reconstituted ATPase was 30 μ M. The proton pumping of reconstituted vesicles could be discharged rapidly by p -trifluoromethoxyphenyl hydrazone (FCCP), hexokinase and vanadate. The hydrolysis of Mg-ATP by both native and reconstituted ATPases obeyed simple Michaelis-Menten plots with a K_m between 0.5 and 0.6 m M. The reconstituted ATPase retained a pH profile similar to that of native enzyme with a maximum of pH 6.5.     

Characterization of a P-type Na+-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum

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(+).