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Purification and Reconstitution of the Vacuolar H+-ATPases from Lemon Fruits and Epicotyls

1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês

10.1074/jbc.272.19.12762

1083-351X

Mathias L. Müller, Ursula Irkens-Kiesecker, Detlef Kramer, Lincoln Taiz,

Seed Germination and Physiology

The vacuolar H+-ATPases (V-ATPases) of lemon fruits and epicotyls were detergent-solubilized, purified by column chromatography, and reconstituted into artificial proteoliposomes. During purification, a vanadate- and nitrate-sensitive ATPase activity, consisting of partially disassembled V-ATPase complexes, was resolved from the V-ATPase peak. ATPase and H+-transport activities of the purified, reconstituted V-ATPases of both fruit and epicotyl exhibited similar inhibitor profiles, except that the fruit V-ATPase retained partial vanadate sensitivity. Since the V-ATPase activity of native fruit tonoplast vesicles is insensitive to inhibitors (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916–1924), membrane lipids or other factors may protect the fruit V-ATPase from inactivation in vivo. A kinetic analysis of H+-pumping and H+-leakage indicated that the reconstituted epicotyl V-ATPase exhibited twice as much intrinsic uncoupling or slip as the reconstituted fruit V-ATPase. Comparison of their subunit compositions by SDS-polyacrylamide gel electrophoresis indicated that the reconstituted fruit V-ATPase is enriched in two polypeptides of 33/34 and 16 kDa. Moreover, the stalks of negatively stained juice sac V-ATPases appeared thicker than those of epicotyl V-ATPases in electron micrographs. The vacuolar H+-ATPases (V-ATPases) of lemon fruits and epicotyls were detergent-solubilized, purified by column chromatography, and reconstituted into artificial proteoliposomes. During purification, a vanadate- and nitrate-sensitive ATPase activity, consisting of partially disassembled V-ATPase complexes, was resolved from the V-ATPase peak. ATPase and H+-transport activities of the purified, reconstituted V-ATPases of both fruit and epicotyl exhibited similar inhibitor profiles, except that the fruit V-ATPase retained partial vanadate sensitivity. Since the V-ATPase activity of native fruit tonoplast vesicles is insensitive to inhibitors (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916–1924), membrane lipids or other factors may protect the fruit V-ATPase from inactivation in vivo. A kinetic analysis of H+-pumping and H+-leakage indicated that the reconstituted epicotyl V-ATPase exhibited twice as much intrinsic uncoupling or slip as the reconstituted fruit V-ATPase. Comparison of their subunit compositions by SDS-polyacrylamide gel electrophoresis indicated that the reconstituted fruit V-ATPase is enriched in two polypeptides of 33/34 and 16 kDa. Moreover, the stalks of negatively stained juice sac V-ATPases appeared thicker than those of epicotyl V-ATPases in electron micrographs. The juice sacs of lemon fruits contain cells that can acidify their vacuoles to as low as pH 2.2 (1Sinclair W.B. The Biochemistry and Physiology of the Lemon and Other Citrus Fruits. University of California, Division of Agriculture and Natural Resources, Oakland, CA1984: 109-156Google Scholar). In contrast, the vacuoles of the surrounding fruit tissues as well as those of vegetative tissues are maintained in the typical vacuolar pH range, 5.0–6.0. The occurrence in lemon of two types of vacuoles with vastly different lumenal pH values provides a convenient experimental system to probe the mechanisms underlying the control of steady state vacuolar pH. One hypothesis to explain the extreme acidity of the juice sac vacuoles is that their H+-ATPase (V-ATPase) 1The abbreviations used are: V-ATPase, vacuolar H+-ATPase; ACMA, 9-amino-6-chloro-2-methoxy-acridine; BCA, bicinchoninic acid; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; DTT, dithiothreitol; Mes, 2-(N-morpholino)ethanesulfonic acid; MOPS; 3-(N-morpholino)propanesulfonic acid; n, H+/ATP stoichiometry; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; QB, Econo-Q running buffer; RB, resuspension buffer; PAGE, polyacrylamide gel electrophoresis. is a functionally specialized isoform capable of generating a greater pH gradient than vegetative V-ATPases. In an earlier report (2Müller M.L. Irkens-Kiesecker U. Rubinstein B. Taiz L. J. Biol. Chem. 1996; 271: 1916-1924Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), we compared the ATP-driven H+-pumping activities of tonoplast-enriched membrane vesicles isolated from juice sacs and seedling epicotyls. In native vesicles, the juice sac V-ATPase generated a steeper proton gradient than the V-ATPase of epicotyls. However, since the epicotyl tonoplast was more permeable to protons than the juice sac tonoplast, the steeper ΔpH generated by the juice sac V-ATPase may have resulted from differences in membrane permeability rather than from intrinsic properties of the pumps. On the other hand, the two H+-pumping activities differed with respect to several kinetic parameters. The epicotyl activity showed a typical V-ATPase profile with respect to ions and inhibitors (i.e.stimulation by chloride, inhibition by nitrate, bafilomycin A1, and N-ethylmaleimide (NEM), and insensitivity to vanadate). In contrast, the proton pumping activity of juice sac tonoplasts was insensitive to nitrate, bafilomycin, and NEM, and was partially inhibited by vanadate. Sensitivity of the juice sac ATPase activity to nitrate and NEM increased following detergent treatment, consistent with the juice sac proton pump's identity as a V-ATPase. However, evidence for the possible existence of a second H+-ATPase on the juice sac tonoplast was also obtained. In nitrate-induced V1-dissociation experiments, the epicotyl vacuolar H+-pumping activity became inactivated with the release of the catalytic subunit from the membrane. Despite the loss of a major portion of the catalytic subunit, the juice sac membranes retained 100% of their H+-pumping activity following nitrate treatment, although vanadate sensitivity increased (2Müller M.L. Irkens-Kiesecker U. Rubinstein B. Taiz L. J. Biol. Chem. 1996; 271: 1916-1924Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Solubilization of the fruit membranes withn-dodecyl-β-d-maltoside and centrifugation on a glycerol gradient resulted in the resolution of two peaks of ATPase activity. The denser of the two peaks was strongly inhibited by nitrate, partially inhibited by vanadate, and exhibited a typical V-ATPase subunit composition on SDS-PAGE gels. The second peak was a vanadate- and nitrate-sensitive ATPase of unknown identity (2Müller M.L. Irkens-Kiesecker U. Rubinstein B. Taiz L. J. Biol. Chem. 1996; 271: 1916-1924Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). It appears that the V-ATPases of lemon fruits and epicotyls may be strongly influenced by their native membranes. To compare the two proton pumps in the same membrane environment we have characterized the properties of purified and reconstituted V-ATPases from fruits and epicotyls. In addition, we have compared negatively stained juice sac and epicotyl tonoplast vesicles by electron microscopy. Our results suggest that native tonoplast lipids of the juice sacs play important roles not only in reducing proton permeability, but in protecting the V-ATPase from inactivation by inhibitors. However, the ability to generate a steeper pH gradient appears to be an intrinsic property of the juice sac V-ATPase. The nature of the vanadate-sensitive "second H+-ATPase" remains unresolved, but may represent partially disassembled V-ATPase complexes. Lemon seeds (Citrus limon L. var. Schaub Rough Lemon) were generously supplied by Willits & Newcomb, Inc., Arvin, CA. Lemon fruits (var. Eureka) were harvested from trees on the campus of the University of California, Santa Cruz. Bafilomycin A1 was from Sigma, BCA protein assay reagents were from Pierce, and n-dodecyl-β-d-maltoside was from Calbiochem. Escherichia coli polar lipid extract was purchased from Avanti Polar Lipids; the NanoOrangeTMprotein quantitation kit and 9-amino-6-chloro-2-methoxy-acridine (ACMA) were from Molecular Probes. All bulk chemicals were purchased from Sigma and Fisher. Tonoplast-enriched membranes from lemon fruit juice sacs and epicotyls were prepared as described previously (2Müller M.L. Irkens-Kiesecker U. Rubinstein B. Taiz L. J. Biol. Chem. 1996; 271: 1916-1924Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). All steps were carried out at 4 °C, and the membranes were maintained on ice. Briefly, juice sacs of three lemons were released into 100 ml of cold fruit homogenization buffer (1.5 m MOPS-KOH, pH 8.5, 2.25% polyvinylpyrrolidone-40, 0.75% bovine serum albumin, 7.5 mm EDTA, 2 mm DTT, and 0.1 mmPMSF). They were ground using a mortar and pestle or, alternatively, homogenized with a Waring blender in a 250-ml flask filled to the top with juice sacs and homogenization buffer, and hermetically sealed to avoid oxidation. Epicotyls (40 g, fresh weight) were harvested with a razor blade and homogenized in 150 ml of cold epicotyl homogenization buffer (0.5 m MOPS-KOH, pH 8.5, 1.5% polyvinylpyrrolidone-40, 0.5% bovine serum albumin, 5 mmEDTA, 2 mm DTT, and 0.1 mm PMSF) using a mortar and pestle. Homogenates were filtered through a 0.28-mm nylon mesh and centrifuged at 12,000 × g for 15 min (Sorvall SS-34 rotor) to eliminate cellular debris, nuclei, and plastids. The supernatant was subjected to ultracentrifugation for 60 min at 132,000 × g in a Beckman SW-28 rotor. The microsomal pellet obtained was resuspended in 15 ml of resuspension buffer (RB; 10 mm BTP-Mes, pH 7.0, 20 mm KCl, 1 mmEDTA, 2 mm DTT, and 0.1 mm PMSF) and further purified on a 10%/35% sucrose step gradient made up in 10 mm BTP-Mes, pH 7.0, 10% glycerol, 20 mm KCl, 1 mm EDTA, 2 mm DTT, and 0.1 mm PMSF. After 60 min of centrifugation at 132,000 × g in a Beckman SW-28.1 rotor, the 10%/35% interface containing tonoplast-enriched membranes was recovered, diluted with RB, and pelleted for 20 min at 174,000 × g in a Beckman TLA-100.3 rotor. The tonoplast-enriched membranes were resuspended in RB at a final concentration of 10 μg of membrane protein/μl. In experiments involving inhibition by NEM, the membranes were resuspended in RB in the absence of DTT. Tonoplast-enriched membranes were made up to 6 mg of protein/ml with RB and solubilized as described previously (2Müller M.L. Irkens-Kiesecker U. Rubinstein B. Taiz L. J. Biol. Chem. 1996; 271: 1916-1924Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) with an equal volume of 4% (w/w)n-dodecyl-β-d-maltoside or 5% (w/w) octyl-β-glucoside in solubilization buffer (10 mmBTP-Mes, pH 7.6, 10% glycerol, 1 mm EDTA, 8 mmMgSO4, 50 mm DTT, 200 μg/mll-α-phosphatidylcholine liposomes, and 0.012% butylated hydroxytoluene). The solubilized membrane proteins were centrifuged for 15 min at 412,000 × g in a Beckman TLA-100.3 rotor, and the supernatant was loaded on a 100 × 1-cm Sephacryl S-400 HR (Pharmacia) chromatography column equilibrated in running buffer (10 mm Tris-Mes, pH 7.0, 0.3% (w/w) Triton X-100, 100 μg/mll-α-phosphatidylcholine, 10% glycerol, 1 mm EDTA, 4 mm MgCl2, 5 mm DTT, and 50 μm PMSF). The column was eluted at 4.5 ml/h with running buffer. 1.5-ml fractions were collected and assayed for ATPase activity in the presence or absence of vanadate and nitrate. After Sephacryl S-400 HR chromatography, fractions making up the leading half of the V-ATPase activity peak were pooled and further purified by Econo-Q anion exchange (Bio-Rad). The proteins were loaded at 0.5 ml/min on a 5-ml column equilibrated with Q buffer (QB; 5 mm Tris-HCl pH 6.0, 50 μg/ml [l-α-phosphatidylcholine, 10% glycerol, 1 mm EDTA, 4 mm MgCl2, 5 mm DTT, and 50 μm PMSF) and eluted with a linear gradient of 0–0.3 m KCl. 1-ml fractions were collected and assayed for ATPase activity in the presence or absence of vanadate and nitrate. Both the lemon fruit and epicotyl preparations showed an activity peak eluting at ∼0.1 m KCl. Additionally, the fruit preparation exhibited an equally nitrate- and vanadate-sensitive activity peak eluting at ∼0.065 m KCl. The fractions making up the peak eluting at 0.1 m KCl were diluted three times with QB, loaded again on an Econo-Q column, and subjected to a second gradient elution. For reconstitution experiments, ATPases bound to this second Econo-Q column were eluted with a 0.2m KCl step to concentrate the activity in one fraction. For chromatography elution buffers and ATPase assays, l-α-phosphatidylcholine type IV-S (Sigma) was dissolved to 10 mg/ml in a total volume of 10 ml of diethyl ether, evaporated to dryness under a stream of nitrogen, lyophilized, resuspended in 10 ml of water, and sonicated to clarity with a Braun-Sonic U probe sonicator. For reconstitution experiments, 5 ml of a 20 mg/ml E. coli polar lipid extract in chloroform (Avanti Polar Lipids) was evaporated to dryness, resuspended together with 17.6 mg of cholesterol in 2 ml of diethyl ether, evaporated to dryness again, lyophilized, resuspended in 2.35 ml of RB, and sonicated to clarity. The liposomes (final concentration, 50 mg/ml) were aliquoted, frozen in liquid nitrogen, and stored at −20 °C. Immediately before being used, the liposomes were thawed and sonicated to clarity again. The reconstitution procedure was based on the method of Ward and Sze (3Ward J.M. Sze H. Methods in Plant Cell Biology. Academic Press, San Diego, CA1995: 149-160Google Scholar). 200 μl of 50 mg/ml E. coli/cholesterol liposomes were added to the 0.2 m KCl step eluate of the Econo-Q column containing the partially purified V-ATPase (1-ml fraction, activity 0.08–0.35 μmol·ml−1·min−1). The mixture was incubated on ice for 30 min, frozen in liquid nitrogen, and thawed again. 0.4 g of wet Bio-Beads SM-2 (Bio-Rad) prepared according to Holloway (4Holloway P.W. Anal. Biochem. 1973; 53: 304-308Crossref PubMed Scopus (641) Google Scholar) were added to the mixture, which was incubated for 30 min at room temperature with gentle rocking. The beads were decanted, and the supernatant was recovered and mixed with another 0.4 g of wet Bio-Beads. The same incubation process was repeated, and the supernatant was again recovered. 100 μl of additional E. coli/cholesterol liposomes were added to the reconstitution mixture, which was again allowed to sit for 30 min on ice. The reconstituted proteoliposomes were then diluted to a final volume of 10 ml with RB, incubated for 20 min at room temperature, and centrifuged for 20 min at 174,000 × g (Beckman TLA-100.3). The pellet containing the reconstituted vesicles was resuspended in 300 μl of RB containing 150 mm KCl. The sizes of the fruit and epicotyl proteoliposomes were determined to be identical by freeze-fracture electron microscopy (data not shown). Proton pumping by tonoplast vesicles and reconstituted proteoliposomes was monitored by quinacrine or ACMA fluorescence quenching. The reaction mix contained 10 mmBTP-Mes, pH 7.0, 250 mm sorbitol, 100 mm KCl, 1 mm azide, 250 nm valinomycin, 2.5 mm ATP, and either quinacrine (10 μm) or ACMA (1.5 μm). For tonoplast-enriched vesicles, 50 μm vanadate was included in the mix. 100 μg of tonoplast-enriched membrane protein or 0.4–1.0 μg of reconstituted proteoliposomes were typically used, and the reaction was started with 4.5 mm MgSO4. Fluorescence quenching (quinacrine: 423 nm excitation, 502 nm emission wave lengths; ACMA: 430 nm excitation, 500 nm emission) was measured in a Perkin-Elmer LS-5 fluorescence spectrophotometer (Perkin-Elmer Corp.). Uncoupling or "slip" rates were estimated based on a kinetic model described by Tu et al. (7Tu S.-I. Nagahashi G. Brouillette J.N. Arch. Biochem. Biophys. 1987; 256: 625-637Crossref PubMed Scopus (23) Google Scholar). According to this model, the proton pumping rate at any given time point during the formation of the gradient can be represented by the following equation, dδdt=nR−k1+k2δ(Eq. 1) where dδ/dt and δ represent the proton pumping rate and the net amount of proton transport, respectively;n is the coupling ratio or stoichiometry of the pump;R, the ATP hydrolysis rate, and k 1and k 2 are first order rate constants representing membrane leakage and slip, respectively. The sum ofk 1 and k 2 can be represented by k i. nR is constant, and at steady state, dδ/dt = 0. It follows that kiδs=nR(Eq. 2) where δs is the steady state value of δ. After substituting and integrating ln1−δδs=−kit(Eq. 3) Equation 3 allows one to estimate k i, the combined leakage and slip rate constant, from proton pumping traces. By analogy, the proton leakage rate can be estimated by linearizing the curves obtained after a pH gradient has been generated, and H+-pumping has been inhibited with EDTA. Again, proton leakage is assumed to obey first order kinetics dδdt=−k3δ(Eq. 3) where k 3 represents the leakage rate constant for this portion of the curve. After integration, 1nδδs=−k3t(Eq. 5) The slip rate constant, k 2, is estimated by subtracting k 3 fromk i. ATP hydrolysis measurements were carried out in a reaction mix containing 2.5 mm ATP, 4.5 mmMgSO4, 100 mm KCl, 1 mm azide, 1 mm molybdate, 2 μm gramicidin, and 1 mg/ml sonicated l-α-phosphatidylcholine liposomes in 25 mm BTP-Mes buffer, pH 7.0. The total reaction volume was 500 μl, and the reaction was started by adding the enzyme to the mix. After 30 min at 37 °C, the reaction was stopped by adding 1.25 ml of Fiske and Subbarow (5Fiske C.H. Subbarow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar) reagent. After 30 min at room temperature, absorbance of the samples at 660 nm was measured in a Spectronic Genesis 5 spectrophotometer (Milton Roy, Rochester, NY). Boiled membranes were used for background estimates. Where nitrate-sensitive or vanadate-sensitive activity is reported, the results are expressed as the difference in activity in the presence or absence of 400 mm KNO3 or 400 μmNa3VO4, respectively. Protein concentrations were measured routinely by a modified BCA protein assay (6Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.L. Goeke B.J. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18647) Google Scholar) or with the NanoOrangeTM protein quantitation kit after precipitation of the proteins with cold acetone and delipidation with diethyl ether. SDS-PAGE was according to Laemmli (8Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar) in 12 or 13.5% polyacrylamide gels. The samples were made up in sample buffer to a final concentration of 60 mm Tris-HCl, pH 6.8, 4% SDS, 5% DTT, 10% glycerol, and 0.0125% bromphenol blue. The gels were developed with silver. The electron microscopy experiments were conducted at the laboratory of Prof. Ulrich Lüttge in Darmstadt, Germany. Tonoplast-enriched membranes were pelleted and resuspended at room temperature to a final concentration of ∼1 mg/ml protein in 10 mm potassium phosphate buffer, pH 7.0, containing 5 mm ATP. Negative staining was performed with a solution of 2% methylamine tungstate according to the successive droplet method (9Klink R. Lüttge U. Bot. Acta. 1991; 104: 121-131Crossref Scopus (24) Google Scholar). A 5-μl droplet of membrane suspension was applied to a Formvar-coated 700-mesh/hexagonal grid. After 2 min, the droplet was wicked off with filter paper and replaced with a 5-μl droplet of 2% methylamine tungstate. After 15–20 s the stain was also wicked off, and the grid was allowed to dry. Specimens were examined and photographed with a Zeiss EM902 electron microscope (Carl Zeiss, Oberkochen, Germany) operated at 80 kV in the electron filter mode. As a first step in the purification, detergent-solubilized tonoplast-enriched membranes from epicotyls and fruits were layered onto a Sephacryl S-400 HR column, and the protein and ATPase activities were monitored. The protein distribution and ATPase activity profiles are shown in Fig. 1. An octyl-β-glucoside solubilization resulted in a single peak of ATPase activity corresponding to a molecular mass of about 4,500 kDa for both fruit and epicotyl membranes (Fig. 1, A and B). Analysis of the fractions by SDS-PAGE indicated that the peak fractions were enriched in subunits for the V-ATPase (data not shown). The high molecular mass of the complex indicated that the V-ATPase was migrating as an aggregate. The epicotyl peak was inhibited by nitrate only, whereas the fruit peak was sensitive to both nitrate andvanadate. If n-dodecyl-β-d-maltoside was used to solubilize the membranes, two peaks of ATPase activity were obtained for both fruit and epicotyl membranes, a nitrate-sensitive V-ATPase activity peak, which migrated either as a 4,500-kDa aggregate as in the octy-β-glucoside experiment (Fig. 1 D), or as a lower molecular mass aggregate of about 1,500 kDa (Fig. 1 C), and a second peak with an apparent molecular mass of about 250 kDa (Fig. 1,C and D). The second peak was both nitrate- and vanadate-sensitive. Treatment of the octyl-β-glucoside peak fractions with n-dodecyl-β-d-maltoside did not induce the appearance of the second peak (data not shown). Hence the second peak is not a degradation product of the first peak, but appears to be specifically solubilized from the membrane byn-dodecyl-β-d-maltoside. The Sephacryl S-400 HR V-ATPase peak fractions from then-dodecyl-β-d-maltoside solubilization were further purified on two successive Econo-Q anion exchange columns. After a first passage over the column, both the fruit and the epicotyl V-ATPases showed an activity peak eluting at 0.1 m KCl. This peak was nitrate-sensitive and vanadate insensitive in the epicotyl preparation, and nitrate-sensitive and partially vanadate-sensitive in the case of the fruit. In addition, the fruit preparation exhibited a second peak of activity at 0.065 mKCl that was inhibited equally by nitrate and vanadate (data not shown). Both the single epicotyl ATPase activity peak and the fruit peak eluting at 0.1 m KCl contained typical V-ATPase subunits when analyzed by SDS-PAGE. The nitrate- and vanadate-sensitive fruit ATPase activity peak eluting at 0.065 m KCl appeared to co-purify with selected V-ATPase subunits rather than with any specific polypeptides. However, the presence of a low abundance contaminant with high ATP hydrolytic activity cannot be ruled out. When the fractions making up the more nitrate-sensitive activity peak were pooled and further purified on a second Econo-Q column, further separation of the vanadate-sensitive from the nitrate-sensitive activities was achieved (Fig. 2). The peak of maximum nitrate-sensitive activity was further enriched in the complete set of V-ATPase subunits and was depleted in contaminating bands, mainly a 100 kDa polypeptide (Fig. 2 B, fraction 40). Bands at 97, 66, 55/56, 52, 42/43, 36, 33, 31, 17, 14, and 13 kDa co-migrated with the peak of activity. Most notably, the doublet at 33/34 kDa was present only in the fruit preparation, and only the 33-kDa component of the doublet co-migrated with the more nitrate-sensitive activity peak. In most experiments, a 16-kDa band also co-migrated with the nitrate-sensitive activity peak, although it appears to be shifted to fraction 42 in the gel of Fig. 2 B. The nitrate- and vanadate-sensitive ATPase activity peak eluting at 0.065 m KCl was enriched in the 97- and 36-kDa bands, and in the 55/56-kDa doublet. The 33/34-kDa doublet was present, but only the 34-kDa component of the doublet co-migrated with the peak of nitrate- and vanadate-sensitive activity. The 66-kDa polypeptide (V-ATPase catalytic or A subunit) was also present, although in reduced amounts compared with the 55/56-kDa doublet (V-ATPase "regulatory" or B subunit). The strong doublet at 25/26 kDa, present in all fractions eluting from both Econo-Q columns, did not show a consistent pattern of co-migration with any of the two activities and is therefore thought to represent a contaminant. The specific activities of the purified V-ATPases (average ± S.D. of four purifications) were 9.5 ± 1.5 μmol of Pi·mg−1·min−1 and 6.9 ± 2.2 μmol Pi·mg−1·min−1 for the epicotyl and fruit, respectively. The sensitivities of the two purified V-ATPases to various inhibitors is shown in Fig.3. Both V-ATPases were about equally inhibited by nitrate (Fig. 3 A), bafilomycin (Fig. 3 B), and NEM (Fig. 3 C). In contrast, only the fruit V-ATPase showed partial inhibition by vanadate (Fig. 3 D). Fig. 3 Dalso shows that the fractions making up the V-ATPase peak after S-400 HR chromatography progressively lost their vanadate sensitivity with subsequent Econo-Q column purifications. This indicates either that the vanadate sensitivity is associated with a contaminating ATPase, or that a subpopulation of V-ATPases, perhaps partial V1 complexes, exhibit nitrate- and vanadate-sensitive ATPase activity. The latter hypothesis is supported by the Econo-Q activity profiles and gels in which the main vanadate-sensitive peak exhibited a subunit composition compatible with that of a V1 complex partially depleted of its catalytic subunit (Fig. 2). Partially purified fruit and epicotyl V-ATPases from a step elution of the second Econo-Q column (see "Experimental Procedures") were reconstituted into artificial proteoliposomes, and their proton pumping activities were compared. When the proton gradients had stabilized, the reactions were stopped by adding EDTA, allowing the pH gradients to collapse due to proton leakage. In Fig. 4, the upper panel shows six different comparisons based on five different experiments (an epicotyl trace is shown twice in B and E, and a fruit trace is shown twice in A and B). Panels A and C represent equal protein concentrations. The proteoliposome concentrations in Fig. 4, B and C, were chosen to give equal initial rates of proton pumping. In Fig. 4,D and E, the protein concentrations were normalized to generate equal fluorescence quenching at equilibrium. In Fig. 4 E the proteoliposomes also displayed equal proton leakage rates. Fig. 4 F represents aged proteoliposomes with leaky membranes. When equal protein concentrations of freshly prepared proteoliposomes were used (Fig. 4, A and C) or when the concentrations of reconstituted fruit and epicotyl proteoliposomes were adjusted to yield equal initial rates of proton pumping (Fig. 4,B and C), the reconstituted fruit V-ATPase consistently generated a steeper pH gradient than the reconstituted epicotyl enzyme. When the proteoliposome concentrations were adjusted so as to build up equal pH gradients at equilibrium, the initial rate of pumping by the fruit V-ATPase was lower than that by the epicotyl enzyme (Fig. 4 D). This latter result was obtained even when the proteoliposomes exhibited equal leakage rates (Fig.4 E). Leakage and intrinsic uncoupling or "slip" rates were estimated according to Tu et al. (7Tu S.-I. Nagahashi G. Brouillette J.N. Arch. Biochem. Biophys. 1987; 256: 625-637Crossref PubMed Scopus (23) Google Scholar) as detailed under "Experimental Procedures." For each of the curves shown in Fig. 4, A–F, the rate constants k i, k 3, and k 2 are given in the table in the lower panel. If all five fruit and epicotyl traces are considered, the average leakage rate constants for the fruit and epicotyl proteoliposomes are approximately equal, 0.047 ± 0.029 and 0.045 ± 0.011, respectively. In contrast, the slip rate constants average 0.341 ± 0.121 for the fruit V-ATPase, and 0.687 ± 0.212 for the epicotyl enzyme. The average epicotyl/fruit slip ratio is 2.0 ± 0.3. These values were obtained by considering the initial third of the proton pumping curves and the second half of the leakage curves. If the entire curves were included in the calculations, the epicotyl/fruit slip ratio averaged 2.4 ± 0.4. Note that the slip rate of the reconstituted epicotyl V-ATPase was higher than that of the fruit enzyme under every condition tested. The reconstituted proteoliposomes containing purified fruit and epicotyl V-ATPases exhibited similar polypeptide profiles, as shown in Fig. 5. Both had bands at 66, 55/56, 52, 42, 36, 31, 17, 16, and 13 kDa. In addition, the fruit V-ATPase contained bands at 100 and 78 kDa, as well as a doublet at 33/34 kDa. The 100- and 78-kDa bands by themselves had no ATPase activity as shown by the second Econo-Q profile (Fig. 2). Quantitative differences were also observed. For example, the fruit enzyme was strongly enriched in a 16-kDa polypeptide, it was slightly depleted in the catalytic subunit (66 kDa), compared with the epicotyl, and had a more pronounced doublet at 55/56 kDa. Table I shows the sensitivities of the two reconstituted proton pumps to nitrate, bafilomycin, and NEM. Proton pumping by the reconstituted fruit V-ATPase was slightly less sensitive to nitrate and NEM than the epicotyl V-ATPase, especially at low concentrations, but it was as sensitive as the epicotyl V-ATPase to bafilomycin. The fruit V-ATPase also retained its partial sensitivity to vanadate (Fig.6 A). Because the fruit proteoliposomes were 100% sensitive to low concentrations of bafilomycin, the vanadate sensitivity of the pump cannot be due to a contaminating P-type ATPase (Fig. 6 B). H+-pumping by the reconstituted epicotyl V-ATPase was completely insensitive to vanadate. Furthermore, the fruit V-ATPase, which was insensitive to oxidation in its native membrane (2Müller M.L. Irkens-Kiesecker U. Rubinstein B. Taiz L. J. Biol. Chem. 1996; 271: 1916-1924Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), was now as prone to oxidation as the epicotyl V-ATPase (Fig. 7), and the inhibition could be partially reversed by 50 mm DTT.Table ISensitivity of purified, reconstituted V-ATPases form lemon fruits and epicotyls to various inhibitorsTreatmentActivityTonoplast enriched membranesReconstituted V-ATPaseEpicotylFruitEpicotylFruit%Control100100100100100 mm KNO31 ± 080 ± 10 ± 06 ± 31 nmBafilomycin A127 ± 12103 ± 40 ± 01 ± 1100 μm NEM1 ± 082 ± 03 ± 28 ± 31 mmNaN371 ± 481 ± 189 ± 1399 ± 1Purified V-ATPases, reconstitute