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• W2038565910 abstract "Rotation catalysis theory has been successfully applied to the molecular mechanism of the ATP synthase (F0F1-ATPase) and probably of the vacuolar ATPase. We investigated the ion binding step toEnterococcus hirae Na+-translocating V-ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 ± 1 Na+ bound/enzyme molecule, with a single high affinity (K d(Na+) = 15 ± 5 μm). The number of cation binding sites is consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. Release of the bound22Na+ from purified molecules in a chasing experiment showed two phases: a fast component (about two-thirds of the total amount of bound Na+;k exchange > 1.7 min−1) and a slow component (about one-third of the total;k exchange = 0.16 min−1), which changes to the fast component by adding ATP or ATPγS. This suggested that about two-thirds of the Na+ binding sites of the Na+-ATPase are readily accessible from the aqueous phase and that the slow component is important for the transport reaction. Rotation catalysis theory has been successfully applied to the molecular mechanism of the ATP synthase (F0F1-ATPase) and probably of the vacuolar ATPase. We investigated the ion binding step toEnterococcus hirae Na+-translocating V-ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 ± 1 Na+ bound/enzyme molecule, with a single high affinity (K d(Na+) = 15 ± 5 μm). The number of cation binding sites is consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. Release of the bound22Na+ from purified molecules in a chasing experiment showed two phases: a fast component (about two-thirds of the total amount of bound Na+;k exchange > 1.7 min−1) and a slow component (about one-third of the total;k exchange = 0.16 min−1), which changes to the fast component by adding ATP or ATPγS. This suggested that about two-thirds of the Na+ binding sites of the Na+-ATPase are readily accessible from the aqueous phase and that the slow component is important for the transport reaction. Na+ binding of V-type Na+-ATPase in Enterococcus hirae.Journal of Biological ChemistryVol. 275Issue 28PreviewPage 13417: Table I was not aligned properly. The correct table is shown below . Full-Text PDF Open Access adenosine 5′-O-(3-thiotriphosphate) 5′-adenylylimido-diphosphate N,N′- dicyclohexylcarbodiimide Ion motive ATPases that do not form phosphorylated intermediates are divided into two types: F0F1-ATPase (F-ATPase) and V0V1-type ATPase (V-ATPase). F-ATPase functions as an ATP synthase in mitochondria, chloroplasts, and oxidative bacteria (1.Senior A.E. Annu. Rev. Biophys. Chem. 1990; 19: 7-41Crossref PubMed Scopus (329) Google Scholar). V-ATPase functions as a proton pump in acidic organelles, in plasma membranes of eukaryotic cells (2.Nelson N. Taiz L. Trends. Biochem. Sci. 1989; 14: 113-116Abstract Full Text PDF PubMed Scopus (244) Google Scholar), and in bacteria (3.Gogarten J.P. Kibak H. Dittrich P. Taiz L. Bowman E.J. Bowman B.J. Manolson M.F. Poole R.J. Data T. Oshima T. Konishi J. Denda K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6661-6665Crossref PubMed Scopus (538) Google Scholar). Because V-type and F-type ATPases resemble each other both structurally and functionally, it is accepted that the reaction mechanism and energy coupling mechanism of the two ATPases are similar (2.Nelson N. Taiz L. Trends. Biochem. Sci. 1989; 14: 113-116Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 4.Nelson N. Biochim. Biophys. Acta. 1992; 1100: 109-124Crossref PubMed Scopus (157) Google Scholar, 5.Dimroth P. Wang H. Grabe M. Oster G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4924-4929Crossref PubMed Scopus (135) Google Scholar). The “rotation catalysis” mechanism, proposed by P. D. Boyer (6.Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (928) Google Scholar), has now been verified as the mechanism of F-ATPase; the energy of ATP hydrolysis is converted into the physical force in the form of rotation of the γ subunit, with three ATP hydrolyses/rotation (7.Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1974) Google Scholar, 8.Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (461) Google Scholar, 9.Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (465) Google Scholar, 10.Yasuda R. Noji H. Kinosita K. Yoshida M. Cell. 1998; 93: 1117-1124Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). Therefore, an important question is how the physical rotation and H+ transport are connected. Two different models for the mechanism of ion translocation through the F0 portion have been proposed for the H+-ATPase of Escherichia coli (11.Junge W. Lill H. Engelbrecht S. Trends. Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (443) Google Scholar, 12.Dmitriev O.Y. Altendorf K. Fillingame R.H. Eur. J. Biochem. 1995; 233: 478-483Crossref PubMed Scopus (45) Google Scholar, 13.Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar, 14.Elston T. Wang H. Oster G. Nature. 1998; 391: 510-514Crossref PubMed Scopus (448) Google Scholar) and the Na+-ATPase ofPropionigenium modestum (5.Dimroth P. Wang H. Grabe M. Oster G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4924-4929Crossref PubMed Scopus (135) Google Scholar, 15.Kaim G. Dimroth P. EMBO J. 1998; 17: 5887-5895Crossref PubMed Scopus (72) Google Scholar). To fully understand the transport mechanism, characterization of the ion binding reaction of the F0 portion is essential. However, in both cases, it was difficult to examine the ion binding to the F0 portion directly, because for E. coli the ion was a proton and forP. modestum the affinity was too low (K m= 0.8 mm) (16.Laubinger W. Dimroth P. Biochemistry. 1988; 27: 7531-7537Crossref PubMed Scopus (161) Google Scholar). A V-type ATPase transports Na+ or Li+ in the eubacterium Enterococcus hirae (17.Kakinuma Y. Igarashi K. FEBS Lett. 1990; 271: 97-101Crossref PubMed Scopus (40) Google Scholar). The enzyme consists of nine subunits encoded by a Na+-responsive operon (designated ntp) (18.Takase K. Kakinuma S. Yamato I. Konishi K. Igarashi K. Kakinuma Y. J. Biol. Chem. 1994; 269: 11037-11044Abstract Full Text PDF PubMed Google Scholar, 19.Murata T. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1996; 271: 23661-23666Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The ATPase activity and Na+ uptake activity of purified E. hiraeV-ATPase were absolutely dependent on the presence of Na+(20.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1997; 272: 24885-24890Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 21.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biochem. (Tokyo). 1999; 272: 24885-24890Google Scholar), with K m values for Na+ of 20 μm and 4 mm and a Kt value for Na+ of 40 μm. These lowK m and Kt values enabled measurement of ion binding to this ATPase. In this study, we report the22Na+ binding properties of E. hiraeNa+-ATPase. This is the first direct demonstration of cation binding in the studies of V- and F-ATPases. The E. hirae strains ATCC 9790 (wild type strain) and 25D/pCemtp18, a mutant defective in the production of F0F1-ATPase harboring a plasmid containing the whole Na+-ATPase (ntp) operon (20.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1997; 272: 24885-24890Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), were used in this study. Cells of ATCC 9790 were cultured at 37 °C in KTY medium (1% Bacto-tryptme, 0.5% Bacto-yeast extract, 1% glucose, and 1% K2HPO4) (19.Murata T. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1996; 271: 23661-23666Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Cells of 25D/pCemtp18 were cultured at 37 °C in KTY medium containing 0.5m NaCl supplemented with 10 μg/ml erythromycin. Membrane vesicles were prepared as described previously (20.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1997; 272: 24885-24890Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Purification of Na+-ATPase was performed using anion exchange and gel filtration chromatographies as described previously (21.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biochem. (Tokyo). 1999; 272: 24885-24890Google Scholar). Concentration of the purified sample was carried out using ultrafiltration (Amicon, YM 100 filter). The incubation mixture contained 0.1 mg of protein of membrane vesicles in 50 μl of A buffer (50 mm Tris-HCl, 5 mm MgCl2, 20% glycerol, 1 mmdithiothreitol, pH 7.5) and 22NaCl (final concentration, 20 μm; 35,000 cpm/nmol). The concentration of contaminating Na+ in the mixture before addition of 20 μm22NaCl was measured using flame photometry. The mixtures including the membrane vesicles from ATCC 9790 and 25D/pCemtp18 had Na+ concentrations of 4 and 8 μm, respectively. We took into account the contaminating Na+ to estimate the total concentration of Na+ in the mixture. The mixture was incubated for 60 min at room temperature; this period was sufficient to saturate the Na+ binding sites of the membrane (data not shown). The mixture was applied to a spin column of 400 μl Dowex-50 WX4–200 (H+ form, Sigma) preloaded with K+, and the free 22Na+ was quickly separated from bound Na+ on membrane vesicles by centrifuging the spin column at 10,000 × g for 5 s at room temperature and washing the column once with 200 μl of A buffer (Dowex-50 method). The eluted fractions from the column were combined. The radioactivities in the eluted and trapped fractions were determined using a γ-scintillation counter (Aloka, Tokyo). The amount of specific binding of Na+ to the membrane vesicles was calculated by subtracting the amount of nonspecific binding that was measured by diluting the radioactivity in the reaction mixture with 10 mm nonradioactive NaCl. In addition to the Dowex-50 method, an ultracentrifugation method was also used (22.Mogi T. Anraku Y. J. Biol. Chem. 1984; 259: 7797-7800Abstract Full Text PDF PubMed Google Scholar). The mixture described above was centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatant was regarded as having only free22Na+. The measurement was repeated five times and averaged, and the standard deviation (S.D.) was calculated. The molecular mass of the Na+-ATPase is estimated as 664 kDa, when calculated from the molecular mass of each subunit and the subunit ratio (20.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1997; 272: 24885-24890Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Thus, 1 mg of the purified enzyme corresponds to 1.5 nmol. The incubation mixture contained 0.02 mg of protein (final concentration, 0.6 μm) of the purified enzyme and 22NaCl (final concentration, 20 μm; 35,000 cpm/nmol) in 50 μl of A buffer plus 0.02% dodecyl maltoside (B buffer). The same mixture, but with various concentrations of 22NaCl, was used for determination of the Na+ concentration dependence of Na+ binding. The concentration of contaminating Na+ in the mixture before the addition of 22NaCl was found to be 2 μm by flame photometry. The contaminating Na+was taken into account to estimate the total concentration of Na+ in the mixture. The mixture was incubated for 60 min at room temperature, which was long enough to saturate the Na+binding sites of the purified enzyme. The free22Na+ was quickly separated by the Dowex-50 method using B buffer as the washing solution, and the amount of Na+ bound to the enzyme was calculated as described above. The equilibrium-dialysis method was also applied using a microequilibrium-dialysis apparatus as described previously (23.Yamato I. Rosenbusch J.P. FEBS Lett. 1983; 151: 102-104Crossref PubMed Scopus (13) Google Scholar). The purified enzyme (final concentration, 3 μm ATPase) in 50 μl of B buffer was placed in a chamber, and 22NaCl (final concentration, 20 μm; 35,000 cpm/nmol) in 50 μl of B buffer was placed in another chamber. The concentration of contaminating Na+ in the mixture including 3 μm purified enzyme was found to be 5 μm and was taken into account to estimate the total concentration of Na+. The difference in radioactivities in the two chambers after dialysis for 3 h at 4 °C was used to calculate the specific binding to the purified sample. S.D. was calculated for five repeated experiments. Chasing experiments were started by adding 10 mmnonradioactive NaCl with or without 2 mm ATP (or ATPγS1) to the incubation mixture equilibrated with 20 μm22Na+, and free 22Na+was quickly separated by the Dowex-50 method at various time intervals. The release of 22Na+ bound to the purified enzyme was because of the exchange reaction with nonradioactive Na+; the release of 22Na+ from the enzyme was dependent on the concentration of added nonradioactive Na+ (data not shown). If the Na+ binding to Na+-ATPase is a simple binding reaction, the exchange rate constant (k exchange) should correspond to the dissociation rate constant (k off) of Na+ in the binding reaction. In this paper, we used the empirical k exchange values, because we do not know the detailed mechanism of the Na+ binding reaction. Protein was determined according to the method of Lowry et al. (24.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin used as standard. The Na+-ATPase activity was determined in the presence of 25 mm NaCl as described previously (20.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1997; 272: 24885-24890Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar); the reaction was started by the addition of 2 mm ATP, sometimes after a 10-min preincubation with various reagents. Li+ of AMP-PNP (Sigma) and adenosine ATPγS (Sigma) were replaced with H+ by passing these chemicals through a 10-ml Dowex-50 WX4–200 (H+ form) column. The pH values of the H+ forms of AMP-PNP and ATPγS were adjusted to 7.5 with Tris. 22NaCl (1.36 TBq/mmol) was obtained from NEN Life Science Products. The Na+-ATPase activity in the membrane vesicles prepared from strain 25D/pCemtp18 was 5 μmol of Pi/min/mg of protein. Specific binding of Na+to the same membrane vesicles was 0.8 ± 0.1 nmol/mg of protein when assayed in the presence of 20 μm22Na+. A similar value of Na+binding was obtained using the centrifugation method (0.8 ± 0.1 nmol/mg of protein). Because E. hirae V-ATPase is induced in response to an increase in intracellular Na+ concentration (19.Murata T. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1996; 271: 23661-23666Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), the amount of this enzyme is limited when cultured in Na+-starved KTY medium (19.Murata T. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1996; 271: 23661-23666Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Na+-ATPase activity in the membrane vesicles of 9790 (wild type strain) grown in KTY medium was not detectable; the amount of Na+ binding to the membrane vesicles was 0.04 ± 0.01 nmol/mg of protein. These findings suggest that more than 93% of the amount of Na+binding to membrane vesicles from 25D/pCemtp18 was because of the Na+-ATPase. The Na+-ATPase activity of the purified enzyme was 26 μmol of Pi/min/mg of protein, which represents an ∼5-fold purification from the membrane vesicles, because specific activities of the ATPase in the solubilized and membrane-embedded states were similar (data not shown). Specific binding of Na+ to the purified sample in the presence of 20 μm22Na+ was 4.0 ± 0.5 nmol/mg protein. A similar value (4.2 ± 0.4 nmol/mg protein) was obtained using the equilibrium dialysis method. The specific binding of Na+ to the purified sample was about five times higher than that to the membrane vesicles, which was consistent with the purification efficiency. These findings indicate that the purified Na+-ATPase retained a Na+ binding capacity similar to that in the native state. Fig.1 shows the NaCl concentration dependence of the Na+ binding in purified Na+-ATPase. The Scatchard plot (Fig. 1, inset) of this data has an intercept on the abscissa at 6.2, indicating that about 6 mol (S.D. 6 ± 1) of Na+ bind/mole of enzyme. The slope of the Scatchard plot indicates that the dissociation constant (K d(Na+)) is 15 μm (S.D. 15 ± 5). This value is similar to the higher (20 μm) of the two K m values for Na+ of the ATPase activity (20.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1997; 272: 24885-24890Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 21.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biochem. (Tokyo). 1999; 272: 24885-24890Google Scholar). Nitrate is known to inhibit V-type ATPase probably by dissociating the V1 complex of the ATPase (25.Moriyama Y. Nelson N. J. Biol. Chem. 1987; 262: 9175-9180Abstract Full Text PDF PubMed Google Scholar). This reagent inhibited the Na+-ATPase activity of both the purified sample and the membrane vesicles (Table I, row 2), but it did not affect the Na+ binding of the purified sample or membrane vesicles. This result is consistent with a model in which the ion binding sites reside in the membrane-embedded portions of F-ATPase (5.Dimroth P. Wang H. Grabe M. Oster G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4924-4929Crossref PubMed Scopus (135) Google Scholar, 14.Elston T. Wang H. Oster G. Nature. 1998; 391: 510-514Crossref PubMed Scopus (448) Google Scholar) and V-ATPase (21.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biochem. (Tokyo). 1999; 272: 24885-24890Google Scholar, 26.Arai H. Berne M. Forgac M. J. Biol. Chem. 1987; 262: 11006-11011Abstract Full Text PDF PubMed Google Scholar). Another potent inhibitor of V-type ATPase, destruxin B (27.Muroi M. Shiragami N. Takatsuki A. Biochem. Biophys. Res. Commun. 1994; 205: 1358-1365Crossref PubMed Scopus (73) Google Scholar), also had no effect on the Na+ binding, although it inhibited the ATPase activity (Table I, row 3).Table IEffect of various reagents on Na + -ATPase activity and Na + binding capacity of purified samples and membrane vesicles (25D/pCemtp18)ReagentPurified sampleMembrane vesicles (25D/pCemtp18)Relative Na+-ATPase activityRelative binding capacityRelative Na+-ATPase activityRelative binding capacity%%%%1No addition100100100100250 mm KNO32295 ± 32695 ± 930.1 mm Destruxin B1896 ± 52097 ± 640.1 mm DCCD (−25 mm NaCl)1548 ± 84080 ± 551 mm DCCD (−25 mm NaCl)214 ± 8340 ± 1061 mm DCCD (+25 mm NaCl)8990 ± 59092 ± 572 mm AMP-PNP1692 ± 72296 ± 782 mm ATPγS396 ± 7893 ± 592 mm ADP7293 ± 676100 ± 4102 mm ADP, 2 mm Pi7098 ± 47296 ± 8Na+ binding was determined by the Dowex-50 method after a 30-min incubation of the mixture with the reagents at the concentrations indicated except for DCCD. 0.1 or 1 mm DCCD was added to the reaction mixture. The mixture was incubated for 10 min in the presence or absence of 25 mm NaCl and was dialyzed against the respective standard reaction mixtures to remove DCCD and Na+. 100% activity corresponds to the control values for ATPase activity and binding capacity under “No addition”. Open table in a new tab Na+ binding was determined by the Dowex-50 method after a 30-min incubation of the mixture with the reagents at the concentrations indicated except for DCCD. 0.1 or 1 mm DCCD was added to the reaction mixture. The mixture was incubated for 10 min in the presence or absence of 25 mm NaCl and was dialyzed against the respective standard reaction mixtures to remove DCCD and Na+. 100% activity corresponds to the control values for ATPase activity and binding capacity under “No addition”. DCCD strongly inhibits F- and V-ATPases by covalently reacting with an acidic amino acid residue of the proteolipid of these ATPases (26.Arai H. Berne M. Forgac M. J. Biol. Chem. 1987; 262: 11006-11011Abstract Full Text PDF PubMed Google Scholar, 28.Hermolin J. Fillingame R.H. J. Biol. Chem. 1989; 264: 3896-3903Abstract Full Text PDF PubMed Google Scholar). Because the coupling ion, Na+, prevented the inhibition by DCCD (21.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biochem. (Tokyo). 1999; 272: 24885-24890Google Scholar), the reactive amino acid was considered to reside at the ion binding site of the F0 or V0portion. Preincubation with the purified sample (0.6 μm) and 0.1 mm DCCD in the absence of Na+ lowered the Na+ binding capacity to about half of its normal level, whereas about 85% of the Na+-ATPase activity was inhibited (Table I, row 4). By preincubation with a large excess of DCCD (1 mm), the Na+ binding of the purified enzyme was reduced to 14 ± 8%, and the Na+-ATPase activity was reduced to 2% (Table I, row 5). In the presence of 0.1 or 1 mm DCCD, the Na+ binding of the membrane vesicles was reduced less than that of the purified enzyme (Table I, rows 4 and 5). As expected, neither the Na+ binding nor Na+-ATPase activity of the purified sample or membrane vesicles was inhibited by preincubation with 1 mm DCCD in the presence of 25 mm Na+ (Table I, row 6). This suggests that the Na+ binding sites are near the DCCD reactive sites in the V0 portion, in agreement with previous data (21.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biochem. (Tokyo). 1999; 272: 24885-24890Google Scholar). The ATPase activity of this enzyme was strongly inhibited by AMP-PNP (K i = 400 μm) (Table I, row 7) and ATPγS (K i = 80 μm) (Table I, row 8). These two ATP analogs are considered to bind to the ATP binding site of F1-ATPase (29.Abrahams J. Leslie A. Lutter R. Walker J. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2764) Google Scholar) and probably bind to the ATP binding site of V1-ATPase. Therefore, these reagents may affect the Na+ binding properties of the enzyme. However, they did not have much effect on the Na+binding capacity (Table I, rows 7 and 8). Neither 2 mm ADP nor 2 mm ADP plus Piaffected the Na+ binding capacity (Table I, rows 9 and 10). This is consistent with a previous report that the V-ATPase reaction was not inhibited by the reaction products ADP and Pi (30.Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (480) Google Scholar). Fig.2 A shows the time course of release of 22Na+ bound to the purified enzyme following the addition of a large excess of nonradioactive NaCl. Semilogarithmic plots of the data appear to show three release phases and exchange rate constants (k exchange): a fast component (about 60% of the total amount of bound Na+;k exchange > 1.7 min−1), a slow component (about 30% of the total; k exchange = 0.16 min−1), and the slowest component (about 10% of the total; k exchange = 0.05 min−1) (Fig. 2 B). The observed fast exchange rate suggests that about 60% of the bound Na+ is freely exchangeable with Na+ in the solution. When 10 mm LiCl instead of NaCl solution was used in a chasing experiment, a similar time course of 22Na+ release and similark exchange values were obtained. However, when K+ or Rb+ was used instead of Na+, essentially no release was observed (data not shown). The cation specificity of chasing was the same as that of stimulation of the transport reaction (20.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1997; 272: 24885-24890Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 21.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biochem. (Tokyo). 1999; 272: 24885-24890Google Scholar). This suggests that the binding of Na+ is related to the transport reaction. When the exchange reaction was assayed with 10 mmnonradioactive NaCl solution containing 2 mm ATP, the slow component (k exchange = 0.16 min−1) disappeared and most of the bound Na+ quickly exchanged (k exchange > 4.8 min−1). Because ions were being translocated in the presence of ATP, the bound Na+ involved in the enzyme reaction of Na+-ATPase should be easily exchanged with nonradioactive Na+ in the solution. Even under these conditions, the slowest component (k exchange = 0.05 min−1) was still present (Fig. 2, C andD), indicating that this slowest component was not related to the enzyme reaction of Na+-ATPase. ATPγS inhibited the ATPase activity but not the Na+binding capacity of the enzyme (Table I, row 8). Interestingly, when 2 mm ATPγS was used in place of ATP in the chasing experiment, most of the bound Na+ quickly exchanged (k exchange > 4.7 min−1), although the slowest component (k exchange = 0.05 min−1) remained, as it did in the case when ATP was added (data not shown). This suggests that the conformational change brought about by binding of ATPγS at the ATP binding site in the V1 portion affects the ion binding properties of the V0 portion, resulting in the disappearance of the slow component (k exchange = 0.16 min−1) of the exchange rates. The exchange rate constant (k exchange) of Na+ bound to DCCD-treated Na+-ATPase was 0.05 min−1 (Fig. 2, E and F), corresponding to that of the slowest component, which was not related to the enzyme reaction of the Na+-ATPase. This suggests that all the Na+ binding sites of the purified enzyme that are involved in Na+ transport reaction react with DCCD. In this study, the 22Na+ binding of a V-type Na+-ATPase (that of E. hirae) was measured. This is the first direct demonstration of cation binding in the studies of V- and F-ATPases. Several of the properties of the Na+ binding of this enzyme are similar to those that previously reported for the enzyme's ATPase activity and transport activity (20.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1997; 272: 24885-24890Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 21.Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biochem. (Tokyo). 1999; 272: 24885-24890Google Scholar). These include the cation specificity, the affinity for Na+, and the reactivity to DCCD. Therefore we concluded that the Na+ binding we measured in this study is involved in the transport reaction of the Na+-ATPase. The number of bound Na+/ATPase was estimated as 6 ± 1. With the addition of 10 mm nonradioactive NaCl, about two-thirds of the bound Na+ was released quickly, and the remainder was released more slowly (Figs. 1 and 2). In the rotating model of F-type ATPase, the F0 portion is thought to contain 12 proteolipid 8-kDa subunits, called the c subunits, that form a rotor ring (31.Singh S. Turina P. Bustamante C. Keller D.J. Capaldi R. FEBS Lett. 1996; 397: 30-34Crossref PubMed Scopus (105) Google Scholar) and each is postulated to have one H+ binding site (32.Miller M.J. Oldenburg M. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4900-4904Crossref PubMed Scopus (126) Google Scholar). The proteolipid (NtpK) ofE. hirae V-type ATPase is about 16 kDa and is considered to have one cation binding site (Glu-139). The Na+ transport activity disappeared in the strain with the E139N mutation, which was induced by site-directed mutagenesis (33.Takase K. Yamato I. Igarashi K. Kakinuma Y. Biosci. Biotechnol. Biochem. 1999; 63: 1125-1129Crossref PubMed Scopus (7) Google Scholar). In this study, the number of Na+ binding sites was found to be 6 ± 1. This suggests that the V0 portion of the Na+-ATPase contains six proteolipid (NtpK) subunits. Consistent with this estimate, the number of H+-conducting proteolipid subunits is thought to be six/V-ATPase (26.Arai H. Berne M. Forgac M. J. Biol. Chem. 1987; 262: 11006-11011Abstract Full Text PDF PubMed Google Scholar, 34.Pali T. Finbow M.E. Holzenburg A. Findlay J.B.C. Marsh D. Biochemistry. 1995; 34: 9211-9218Crossref PubMed Scopus (35) Google Scholar). The reason that 0.1 mm DCCD in the absence of Na+ strongly inhibited the ATPase activity of the purified enzyme but had only a moderate effect on Na+ binding capacity (Table I, row 4) is unclear. This discrepancy may be because the ATPase activity is inhibited by only a single DCCD molecule. This has been shown to be the case with an F-ATPase (28.Hermolin J. Fillingame R.H. J. Biol. Chem. 1989; 264: 3896-3903Abstract Full Text PDF PubMed Google Scholar), and so it seems likely that it also occurs with the V-ATPase. Because the other subunits in the Na+-ATPase that are not modified by DCCD should retain a Na+ binding capability, the reduction of binding would be expected to be intermediate. Our finding that about two-thirds of the bound Na+ was released quickly (fast component) and the remainder was released more slowly (slow component) (Fig. 2) suggested that about two-thirds of the Na+ binding sites of the Na+-ATPase are readily accessible from the aqueous phase. The slow component disappeared in the presence of ATP or ATPγS, suggesting that all or most Na+ binding sites of the enzyme are freely accessible from the aqueous phase under these conditions. In addition, the binding of an ATP analog (ATPγS) affected the existence of the slow component of release of the bound Na+. We believe that this indicates that the slow component must be involved in the transport reaction. The present findings concerning the Na+ binding reaction of the V-type Na+-ATPase in E. hirae should be useful in understanding the mechanisms of cation transport through membrane-embedded portions of V- and F-ATPases." @default.
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