substrate preference was ATP > ITP w UTP > GTP >. CTP. The affinity ... marked âadvertisementâ in accordance with 18 U.S.C. Section 1734 solely to indicate ...
THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 262, No. 32, Issue of November 15, pp. 15780-15789,1987 Printed in U.S.A.
0 1987 by The American Society for Biochemistry and Molecular Biology, Inc.
Immunoaffinity Purification and Characterizationof Vacuolar H+ATPase from BovineKidney* (Received for publication, February 19,
1987)
Stephen GluckS and Jerry Caldwell From the Department of Medicine, The University of Chicago, Chicago, Illinois 60637
Vacuolar proton-translocating ATPase from bovine medulla by conventional and HPLC’ chromatography.2 The kidney was purified in one step by immunoprecipita- enzyme is a large molecular weight protein with multiple tion and immunoaffinity chromatography using an im- component polypeptides. The partially purified enzyme was mobilized anti-H+ATPase monoclonal antibody. The used to generate a monoclonal antibody to the bovine kidney monoclonal antibodyaffinity matrix coprecipitated vacuolar proton pump (32).The antibody, H6.1, was able to polypeptides with M, of 70,000, a cluster at 56,000, immunoprecipitate specifically both solubilized NEM-sensi45,000, 42,000, 38,000, 33,000, 31,000, 15,000, tive ATPase activity, andproton-transporting microsomal 14,000,and 12,000 from solubilized bovine kidney vesicles, and, by immunocytochemistry, showed membrane microsomal membranes, a pattern thatwas unaffected staining of acidic intracellular vacuolar compartments idenby different detergent washing conditions. A nearly identical pattern of polypeptides was observed in tified with the weak base probe N-(3-((2-4-dinitrophenyl)(9). Inthis H+ATPasepartially purified by an entirely independ- amino)propyl)-N-(3-aminopropyl-methylamine) paper, we describe purification of the bovine kidney vacuolar ent method. The immunoaffinity purified H+ATPase had reconstitutively activeATP-induced acidification proton pump by a one-stepprocedure on an €36.1 monoclonal and potential generationthat was inhibited by N-eth- antibody affinity column. The immunoaffinity purified enylamaleimide. The purifiedenzyme had specific activ- zyme retains electrogenic ATP-dependent proton transport, ities as high as 3.1 pmol/min/mg protein, dual pH op- and itsproperties are similar to those of the enzyme in kidney microsomal vesicles. The polypeptide composition of the im. tima at 6.5 and 7.2,and a K, for ATP of 150 p ~ The substrate preference wasATP > ITP w UTP > GTP > munoaffinity purified enzyme is similar to that of kidney CTP. The affinity purifiedH+ATPase was stimulated H+ATPase obtained by conventional chromatography. Howby phosphatidyl glycerol > phosphatidyl inositol w ever, the polypeptide composition and enzymatic properties phosphatidyl choline > phosphatidyl serine. The im- of the affinity purified enzyme differ from those reported for munoaffinity purified enzyme did not require monov- other vacuolar H+ATPases. alent anions or cations for activity, and the divalent cation preference for activity wasMn, Mg w Ca > Co EXPERIMENTALPROCEDURES B Sr, Ba. The enzyme was not inhibited by ouabain, Preparation and Solubilization of Membranes-Bovine kidney miazide, or vanadate, but had k, inhibitory concentrations of 22.2 PM for N-ethylmaleimide, 14.9 MM for crosomes from either medulla or cortex tissue were prepared as described (8)except for omission of the sucrose gradient centrifugaNBD-Cl, 4.9 PM for N,N’-dicyclohexylcarbodiimide, tion. l mM phenylmethylsulfonyl fluoride was added, and membranes 13.8 p~ for 4,4’-diisothiocyanatostilbene-2,2’-disul- were stored a t -70 “C until used. Microsomes were solubilized in a fonic acid, and 315 g M for Zn, values close to those for final mixture containing 5 mg/ml microsomal protein, 10 mM Trisinhibition of proton transport in the native vesicles. C1, 1 mM EDTA, 1 mM dithiothreitol, 0.6% CHAPS, 1.5% nonyl The affinity purified kidney enzyme has similaritiesto glucoside, and 10% glycerol, pH 7.0. The mixture was stirred for 10 but also significant differences in structural and en- min at 4 “C and centrifuged at 150,000 X g for 1 h. The clear zymatic properties from those reported for other vac- supernatant contained the solubilized microsomal membranes. In the experiment in Fig. 2, an ammonium sulfate precipitation was used to uolar H+ATPases.
An electrogenic proton translocating ATPase responsible is both for acidification of intracellular membrane compartments of the vacuolar system (1-6) and for urinary acidification (7,8). In a previous paper, we described partial purification of the vacuolar-type proton pump from bovine kidney
* This work was supported by the Otho Sprague Fund, National Institutes of Health Grant AM 34788 and theSearle Scholar program. 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. $Recipient of a Clinician-Scientist Award from the American Heart Association with funds contributed in part by the Chicago Heart Association. To whom correspondence should be addressed: Dept. of Medicine, Washington University School of Medicine, Jewish Hospital of St. Louis, 216 S. Kingshighway Blvd., St. Louis, MO 63110.
enrich for H+ATPase as follows: saturated ammonium sulfate, pH 7.0,final concentration 40%, was added to thesolubilized microsomes at 4 ‘C, and the mixture was centrifuged at 38,000X g for 15 min. Saturated ammonium sulfate was added to the supernatant to a final concentration of 50%, and centrifugation was repeated. The pellet (40-50% interval fraction)was dissolved in 9 ml/g pellet weight of 10 mM imidazole-acetate, 1 mm sodium azide, 1 mM dithiothreitol, 1 mM EGTA, 10% glycerol, 0.1% CHAPS, 0.1% decylglucoside, pH 7.0. A second ammonium sulfate fractionation was done using a 33-50% interval. This procedure gave a 12.6-fold enrichment of NEM-sensitive ATPase activity over the solubilized membranes.’ Monoclonal Antibody Production-Details of the immunization and
The abbreviations used are: HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-lpropanesulfonate, DCCD, N,N‘-dicyclohexylcarbodiimide;decyl glucoside, decyl-P-D-glucopyranoside;EGTA, [ethylenebis(oxyethylenenitri1o)ltetraacetic acid; NEM, N-ethylmaleimide; nonyl glucoside, nonyl-P-D-glucopyranoside; oxonol V, (bis-(3-phenyl-5-oxoisoxazol-4-y1)pentamethine oxonol; PG, phosphatidylglycerol; SDS, sodium dodecyl sulfate; MES, 2-(N-morpholino)ethanesulfonicacid. S. Gluck and J. Caldwell (1987)Am. J. Physiol., in press.
15780
Immunoaffinity Purification screening procedures for production of the anti-H+ATPaseantibody are reported in the preceding article (32). Briefly, mice were immunized with H’ATPase isolated by combined conventional and HPLC fractionation, and hybridoma supernatants were screened initially for binding to theisolated H’ATPase by a “dot-blot” assay. Supernatants positive on this screen were then tested for immunoprecipitation of NEM-sensitive ATPase activityfrom solubilized kidney microsomes. One antibody, H6.1, fulfilled this criterion and was also able to immunoprecipitate proton-transporting vesicles from bovine kidney microsomes. This antibody was used for the studies reported in this paper. Purification of Monoclonul Antibody H6.1-Ascites was collected from Pristane-treated mice injected intraperitoneally with H6.1 hybridoma cells as described (10). A 30-50% saturated ammonium sulfate fraction of ascites was prepared and dialyzed against 20 mM MES, 5 mM sodium azide, pH 6.0. The sample was centrifuged a t 35,000 X g for 15 min, and about 25 mg was applied to a I-ml Mono S column (Pharmacia) equilibrated in the same buffer and eluted with a 0-0.15 M linear NaCl gradient over 50 min in the same buffer a t 1 ml/min. The fractions were initially assayed for ability to immunoprecipitate solubilized NEM-sensitive ATPase activity. Thereafter, the initial large protein peak, identified by the 280 nm of absorbance, was collected. The pooled fractions were adjusted to pH 7.5 with 1 M Tris andapplied to a 1-ml Mono Q column (Pharmacia) equilibrated in 20 mM Tris-C1,5 mM sodium azide, pH 7.5, and eluted with a 0-0.25 M linear NaCl gradient over 50 min a t 1 ml/min. A large peak (Azm)reproducibly had purified H6.1 immunoglobulin. Preparation of Affinity Gel-Purified H6.1 was coupled to protein A-Sepharose by the method of Schneider et al. (11)a t a ratio of 3 mg of antibody/ml of gel, and 15 mM dimethylpimelimidate was used for coupling. The gel was washed and stored in 20 mM Tris, 5 mM azide, pH 7.5 (TA buffer). Immunoprecipitation of H+ATPase-Immunoprecipitations were performed as follows: polyethylene glycol (molecular weight 20,000) was added to the solubilized membranes to a final concentration of 1%.50 p1 of H6.1 A-Sepharose beads were washed once with 1ml of T A buffer with 1%polyethylene glycol and incubated with solubilized membrane fractions for 1 h at 25 “C. The beads were then washed three times with 1 ml of TA buffer with 0.1% CHAPS and 0.1% nonyl glucoside; then three times with the same buffer containing 500 mM NaC1; then three final washes in the same buffer without salt. In the experiments inFigs. 1 and 4C,the bound protein was eluted from the beads with 50 mM glycine, 5 mM sodium azide, 0.1% CHAPS, 0.1% nonyl glucoside, pH 2.5. In the experiments inFig. 1,other detergents were washed in the washing procedure as indicated. Immunoaffinity Isolation of H+ATPase-Purification of the H+ATPase on the monoclonal antibody affinity column was done by a procedure similar to thatused for the immunoprecipitation experiments. A 5-ml column of H6.1-A-Sepharose was washed with T A buffer with 1% polyethylene glycol, and solubilized membrane containing 1%polyethylene glycol wasapplied to thecolumn. The column was washed successively with 10 volumes each of the same series of buffers used in the immunoprecipitation experiments. The column was then eluted with 5 volumes of 50 mM glycine, 5 mM sodium azide, 0.1% CHAPS, 0.1% nonyl glucoside, pH 2.5. Preparation of Solubilized Soybean Phospholipid-Soybean phospholipid (asolectin; Sigma) was washed with acetone and dissolved in ether as described (12). The lipid was dried under nitrogen in a test tube, and 10 mM Tris-C1, 5 mM sodium azide, pH 7.0 was added to yield a final concentration of40mgof lipid/ml. The lipid was sonicated to near clarity under nitrogen, and a final concentrationof I % CHAPS (w/v) was added and stirred at 4 “C for 30 min. The mixture was centrifuged a t 100,000 X g for 1 h and the solubilized supernatant termed “solubilized soybean phospholipid,” was used as indicated. Assay of Immunoprecipitated ATPase Activity-ATPase activity bound to formalin-fixed protein A-bearing staphylococcal beads was assayed as follows: 10 p l of a 10% suspension of staphylococcal beads (Pansorbin; Behring Diagnostics) was used for each assay tube. 5 pg of rabbit anti-mouse IgG (Cooper; Malvern, PA) per 10 p l of staphylococcal beads was added, and the mixture was allowed to stand 15 min a t 22 “C. Beads were centrifuged and washed twice with 10 mM Tris-C1,500 mM NaCl, pH 7.4 (TBS), andresuspended at theoriginal volume in TBS. 5 pg of purified H6.1 antibody was then added per 10 p1 of beads and allowed to stand 1 h a t 22 “C. Beads were washed once with TA buffer with 1%polyethylene glycol and incubated with detergent-solubilized membranes containing 1%polyethylene glycol for 1 h a t 22 “C. The beads were then washed once in 10 times the
of Vacuolar H+ATPase A
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Sol Micros
2 x AmS
B
T
45
L*
d f L
FIG. 1. A, effect of different detergent washes on immunoprecipitated polypeptide pattern. 50 p l of protein A-Sepharose beads with covalently attatched H6.1 monoclonal antibody was incubated either with whole solubilized membranes (Sol Micros), or with membranes partially purified by two 40-50% ammonium sulfate precipitations (2 X AmS) as under “Experimental Procedures.” Beadswere then washed successivelythree times each with detergent solutions without salt, then with 0.5 M NaCI, then without salt. Solutions contained 10 mM Tris-C1, pH 7.5, 5 mM sodium azide, and detergent either 0.1% CHAPS and 0.1% nonyl glucoside (CHAPS), 0.1% Tween-20 (Tween), or 0.5% Triton X-I00 and 0.5% sodium deoxycholate (TritDOC) as indicated. Beads were then extracted with two 100-plaliquots of 50 mM glycine-C1,5 mM azide, pH 2.8. The bead supernatant (3S), bead pellet ( 3 B ) ,and postincubation-solubilized membrane supernatant (7s)were subjected to electrophoresis on a 10% SDS-polyacrylamide gel. The large band seen near the bottom of the gel in each 3E lane represents monoclonal antibody light chain. The gel shows that the pattern of immunoprecipitated polypeptides was not affected by the detergent washing conditions. B, immunoprecipitation with irrelevant monoclonal antibody MOPC21. MOPC2l was coupled to protein A-Sepharose beads under conditions identical to those used for coupling H6.1. 50 p l of beads were used for immunoprecipitation; a protocol identical to thatused above (washed with buffer containing CHAPS and nonyl glucoside) was employed. The control beads did not immunoprecipitate any of the polypeptides binding to the H6.1 beads. A faint light chain band (15)was visible in the 3B lane.
15782
Immunoaffinity Purification of Vacuolar H+ATPase
originalvolume of buffercontaining the finalconcentrationsof reagents usedfor the ATPase assay plus 0.1% CHAPS and 0.1% nonyl glucoside, and resuspended in 50 p1 of the same. For many of the experiments, 50 pg (100 pg/ml) phosphatidyl glycerol was also added to the assays. ATPase Assays-ATPase (total and NEM-sensitive) was assayed as described previously (8). NEM-sensitive activity was assayed as the difference in ATPase activity after 5 min of preincubation with and without1mM NEM. Assayscontained no other inhibitors except as indicatedin the text, and the ionic conditions and buffers used in each assay are indicated inthe text. For the experiments on pH dependence, assays contained 30 mM Bis-Tris-propaneadjusted with gluconic acid or Tris, and 2 mMMg gluconate. For the experiments on ATP concentration dependence (Fig. 7) assays contained 30 mM Bis-Tris-C1, pH 6.5, 5 mM phosphoenolpyruvate, 50pg/ml pyruvatekinase, 6 mM MgCl,, and concentrations of ATP indicated. For the nucleotide and lipid dependence of activity, and effectsof inhibitors, assays contained 30 mM Bis-Tris-C1, pH 6.5, and 6 mM MgC12. Transport Assays-Assaysfor ATP-dependent proton transport using acridineorange were asdescribedpreviously (8). Transport assays contained 10 mM Bis-Tris-C1, pH 6.5, 150 mM KC1, 6 mM MgC12, and 1 p~ valinomycin; transport was initiated by addition of 1 mM ATP. ATP-dependent potential generation was assayed with the anionic potential-sensitiveprobe oxonol V (20 p ~ using ) a dual wavelength spectrophotometer measuring the 590-625 nm absorbance difference as described (13).Transport buffer contained 10mM Bis-Tris-C1, pH 6.5, 150mM KCl, 6 mM MgC1, and was initiated by addition of 1 mM ATP. Reconstitution of Proton Transport by Immunoprecipitated H+ATPase-50 pl of H6.1-protein A-Sepharose beads were incubated with 1ml of solubilized membranes as described for the immunoprecipitation experiments. Beadswere washed twice with 1 ml of 10 mM Tris-C1,5mM sodium azide, 0.1% CHAPS, 0.1% nonyl glucoside, pH 7.0, the supernatant was removed, and 50 p1 of solubilized soybean phospholipid (40 mg/ml) was added. 1 ml of 10 mM Tris-C1, 10 mM MES, 150 mM KCl, pH 7.0, was added to allow liposome formation by dilution; a final concentration of 2 mM MgC12 was then added. The entire samplewas transferred to a cuvette. Foracidification assays, 10 prn of acridine orange and 1 p~ valinomycin were added; for potential generation assays,5 p M oxonol V was added. Transport was then measured as described above. PolyacrylamideGelElectrophoresis-Discontinuous SDS-polyacrylamide gel electrophoresis was used, and gelswere stained with Coomassie Blue (14). Protein Assays-All protein determinationswere by the method of Lowry etal. (15). The results did not differsubstantiallywhen compared with measurements made with the Bradford method, but the Lowry method allowed measurements of samples containing8 M urea. The amount of solubilizedmembraneproteinboundto the beads was measured by removal with an 8 M urea wash; the protein contained in an identical wash from control beads not incubated with the membrane proteinwas then subtracted. Materials-Polyethylene glycol (M, 20,000) was from Fisher, nonyl and decyl glucoside were from Behring Diagnostics, and protein ASepharosewas from Pierce ChemicalCo. All purified lipids were from AvantiPolarLipids,Inc.(Birmingham,AL).Oxonol V wasfrom Molecular Probes (Junction City, OR). All other reagents were from Sigma, unless otherwise indicated.
nents of the intact enzyme, or intimately associated with it. The number of protein chains bound to the affinity beads should depend on the strengthof the detergent present.Low strength detergentswould not be likely t o dissociate subunits of a multimeric protein but might not be strong enough to dissociate all“nonspecifically” bound proteins. High strength detergents should remove most nonspecifically bound polypeptides but might dissociate actual subunits of the protein as well. Thus, finding that the same group of polypeptides were coimmunoprecipitated in monoclonal antibody affinity beads after washing with detergents of different strengths would provide strong evidence that the polypeptides were components of a multimeric protein. The monoclonal antibody immunoaffinity strategy has been used to purify other intact, heteromultimeric membrane proteins, such as the T cell receptor (16). The immunoaffinity gel was prepared by covalently coupling monoclonal antibody directed against the proton pump, H6.1, to protein A-Sepharose beads. Bovine kidney microsomal membraneswere solubilized under conditions that preserved proton pump enzymatic activity as assayed by NEMsensitive ATPase activity. The H6.1-Sepharose was used initially in a series of immunoprecipitation experiments to examine the polypeptidecomposition of the solubilized protein binding to the beads and to determine the optimal washing andelutionconditions.Bothcrudeandpartially purified solubilized membranes were incubatedwiththeantibody beads. Washing was then carried out withbuffers containing either CHAPS, Tween-20, or Triton and deoxycholate, to represent, respectively, low, medium, and high “stringency” detergent washing. The antibody beads consistently immunoprecipitated 10 different polypeptides at M, of 70,000, a cluster at -56,000, 45,000, 42,000, 38,000, 33,000, 31,000, 15,000, 14,000, and 12,000. As shown in Fig. lA, thepolypeptide composition was unaffected by the several different detergent washing conditions. Most of the bound proteincould be eluted from the beads underacidic pH conditions, and the protein remaining on the beads had the same polypeptide composition as the eluted protein, except for noncovalently attached light chains seen in some preparations of beads. Thus, under all conditions tested, the monoclonal affinity beads coprecipitated the samegroup of polypeptides. In control experiments, an “irrelevant” monoclonal IgG1, MOPC21, coupled to protein A-Sepharose under identical conditions, didnotimmunoprecipitatethese polypeptides(Fig. IB), showing that nonspecific bindingdidnotaccount for the pattern of polypeptides obtained. The beads were next used to prepare an affinity column. Solubilized membranes were applied to the column, which was then washed extensively and eluted as described under “Experimental Procedures.” The applied flow through and elutedfractions from the column are shown in Fig. 2 for RESULTS solubilized microsomal membranes obtained from eitherkidPolypeptide Composition of Enzyme Isolated by Immunopre- ney medulla or cortex. The pattern of polypeptides eluted from the monoclonal affinity column for each microsomal cipitation and Immunoaffinity Purification-In a preceding paper, the bovine kidney H’ATPase was found to be a large preparation was reproducible and identical to that obtained molecular weight enzyme with multiple different component in the immunoprecipitation experiments. In neither the impolypeptides.* As for all heteromultimericenzymes, this find- munoprecipitation experiments nor the column purification ing raised the question of which polypeptides are actual sub- did the pattern of eluted polypeptides resemble that of the units as opposed to contaminants. One means of addressing solubilized membranes. Thus,again the protein bound to and column did not appear be to the result this issue is to use a second, completely independent method eluted from the affinity of purifying the enzyme. Toward this end, the monoclonal of nonspecific binding. of theaffinity purified The polypeptide composition antibody immunoaffinity purification strategy was adopted. of enzymatically active Since the antibody should bind only to a single epitope on the H+ATPase was next compared to that proton pump from bovine kidney medulla in fractions from protein,then,undertheappropriate washing andelution conditions, all polypeptideswhich copurify should be compo- the Mono P column. Fig. 3 shows SDS-polyacrylamide gels
ImmunoaffinityPurification of Vacuolar H+ATPase
15783
purified fromcortexand medulla were similar,but some differences were consistently present. The most obvious difference was in the relative distribution of polypeptides clus20a tered a t M , -56,000. Although this region on gels often contained four or more different polypeptides, those at M , 116 56,000 and 58,000 were most intense. In theenzyme prepared 9C from medullarymicrosomes, the amount of polypeptide at 58,000, assessed by staining intensity, was equal to orslightly greater than that 56,000, at while in the enzyme isolated from 66 cortex, the polyeptide a t M , 56,000 was usually about twice as intense as that58,000. at The significance of the difference in tissue distribution is uncertain, but the observations suggested the possibility that different isoforms of the proton 4s pump exist which have different -56,000 M , subunits. Some additional evidence to support the existence of different isoforms of the enzyme was provided by the distribution of NEM-sensitive ATPase activity andpolypeptide composition of fractions obtained from the Mono P column. In most x 29 of the separationsperformed, NEM-sensitive ATPaseactivity was partially resolved into two distinct peaks, as shown in L Fig. 3. The polypeptide composition in the-56,000 M , region E varied in the different fractions. As shown, the 56,000 M , polypeptide was present in the fractions of the first peak of ATPaseactivity, whereas the 58,000 M , polypeptidewas present mostly in the fractionsin the second peak of ATPase activity. While notconclusive, these observations are consistent with the notion that at least two different isoforms of the proton pump existwhich differ in composition in the -56,000 M , polypeptide and which are partiallyresolved on the Mono I12 P column. The polypeptide a t M , 45,000 had a peak intensity at Mono P fraction 23 in the second peak of ATPase activity. However, no consistentdifference in content of this polypeptide was noted between enzyme immunoaffinity purified from cortex or medulla. Reconstitution of Proton Transport by Immunoaffinity Purified H’ATPase-In the initial characterization studies of the H6.1 monoclonal antibody, it was found that concentradf . . tions as high as 25 pg/ml (ratio of 1:l for antibody/vesicle FIG. 2. Fractions from the monoclonal immunoaffinity col- protein) had no effect on proton transport in native kidney umn. 80 ml of solubilized membranes were applied to a 10-ml column vesicles. The antibody therefore appearedbetononinhibitory. In order to ascertain that the protein bound to the affinity of protein A-HG.l-Sepharose beads, and the columnwas washed and eluted as described under “Experimental Procedures.” Lane I , solu- beads represented a proton pump, purified ATPase bound to bilized cortex membranes, applied(40 pg); lane 2, cortex membranes, the beads was reconstituted into liposomes directly on the flow through (40 pg); lane 3, solubilized medulla membranes (40 pg); beadsand assayedfor ATP-dependentprotontransport. lune 4, medulla membranes, flow through (40 pg); lane 5,cortex eluate Beads were incubated with solubilized bovine kidney micro(15 pg); lane 6, medulla eluate (15 pg). somal membranes andwashed, and solubilized soybean phospholipid was added. The beads were then diluted 20 volumes both of fractions containing NEM-sensitive ATPase activity from the MonoP column and of the immunoaffinitypurified into KC1 buffer to allow liposome formation and were tested A, the reconstituted enzyme H’ATPase. In Fig. 3A, the column fractions were applied for proton transport. In Fig. 4 was tested for the ability to acidify the interiorof the liposome directly, and in Fig. 3B fractions were concentrated by tri(8).Addition of ATP produced chloroacetic acid precipitation before being applied to thegel. by the acridine orange method an immediate acidification that was inhibited by NEM and Several findings were apparent. The pattern of major polypeptides from the Mono P fractions was nearly identical to collapsed by the electroneutralexchange ionophore nigericin. Addition of 50 pl of solubilized soybean phospholipid to the that of the immunoaffinity purified H’ATPase. Thus, the beads was required for transport activity, which presumably samepattern of polypeptidesemergedwhentwo entirely independent methods of isolation were used, adding support allowed liposome formation on the beads to occur. Fig. 4B liposomes also had ATPtothecontentionthattheserepresentsubunits of the shows that the bound reconstituted dependent potential generation, assayed by uptake of the H’ATPase. Inordertodemonstratethe lower molecular weight polypeptides more clearly,the same fractions from the potential-sensitive probe oxonol V, that was inhibited by Mono P column as shown inFig. 3A were trichloroacetic acid- NEM and collapsed by the conductive protonophore, tetraprecipitated prior to being applied to the gel (Fig. 3B). Al- chlorosalicylanilide. Uptake of the anionic probeindicates though high molecular weight contaminating polypeptides (or that the liposomes generated an inside positive potential. To aggregation) were present in the Mono P column fractions, verify that the reconstitutively active protein had the same the patternof major polypeptides was nearly identicalto that composition as that elutedfrom the immunoaffinity column, protein was eluted from the proton-transporting beads and of protein isolated on the immunoaffinitycolumn. The polypeptidecomposition of enzyme immunoaffinity subjected to SDS-polyacrylamide gel electrophoresis. As 1
2
3
4
5
6
“7
2
r:
Immunoaffinity Purification of
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Vacuolar H+ATPase
B
A
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Mono P Fractions
c I
20
/
I
I
\
I
I
I
I
I
20 11 9 66 0
5
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'0 F
x 45 L
I 29-
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-15 -1 4 a12
FIG.3. Comparison of polypeptide composition of Mono P fractions with immunoaffinity purified H+ATPase.A, 40-pl aliquots of fractions from the Mono P column step of H+ATPase isolation from a preceding paper' were applied to a 10% SDS-polyacrylamide gel. In the last lane, 25 pg of immunoaffinity purified bovine kidney medullary H+ATPase was applied. The major polypeptide bands in the two preparations were nearly identical. The 14,000 M,polypeptide was not seen well in the Mono P fractions on this gel but is apparent in Fig. 38. The affinity purified H+ATPase hadsome minor high molecular weight contaminants which were not seen in the preparation shown in Fig. 3B. The relative distribution of the polypeptides in the 56,000 M , cluster in the Mono P fractions varied through the double peak of NEM-sensitive ATPase activity. The 56,000 M , polypeptide had a peak intensity a t fractions 18 and 19, while the 58,000 M , polypeptide was most intense in fractions22 and 23. B, 150-p1aliquots of Mono P column fractions were precipitated with 150 plof 50%trichloroacetic acid, washed twice with 20 mM Tris-C1, pH 7.5, and applied to a 15% SDS-polyacrylamide gel. In the last two lanes, 20 pg of affinity purified bovine kidney medullary ( M ) and cortex (C) H+ATPase were applied. Minor contaminating bands are apparentin the concentrated Mono P fractions, but themajor polypeptides are present inthe affinity purified H'ATPase. Gel shows the composition of the affinity purified H+ATPase more clearly and reveals the 14,000 M , polypeptide in the Mono P fractions.
shown in Fig. 4C, the polypeptide composition obtained was essentially the same as seen in the protein eluted from the affinity column. Enzymatic Properties of the Zmmunoaffinity Purified H+ATPase-The enzyme bound to theaffinity beads retained NEM-sensitiveATPaseactivity and was used for further characterization of the purified proton pump. Isolation of the proton pump by conventional chromatography required ad-
dition of soybean phospholipid to all of the buffers to maintain activity, presumably to prevent delipidation. Washing the beads three times with 1 ml of TA buffer containing 0.1% CHAPS and0.1% nonyl glucoside and no added lipid resulted in a nearly complete loss of enzymatic activity with no change in thecomposition of polypeptides bound to thebeads, again suggesting that detergent removal of lipid caused loss of enzymatic activity. Therefore, a variety of lipids were tested
Immunoaffinity Purification of Vacuolar H+ATPase A
ATP
ATP
7
WIG
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B
TCS
c Mr x 10"
ATP
d l
L
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y -15 -14 -12
c
It
TdS
1IG
FIG. 4. Reconstitution of proton transport by immunoprecipitated H+ATPase. Immunoprecipitation of H+ATPase from solubilized membranes and formation of liposomes on the beads was as described under "Experimental Procedures." 1 mM ATP was added where indicated. A, transport assays with acridine orange, showing ATP-dependent acidification by the affinity precipitated enzyme. As shown, transport required addition of phospholipid and was completely inhibited by 1 mM NEM. 1 p~ nigericin was added where indicated. B, TransportassayswithoxonolV,showingATP-dependentpotentialgeneration. Signalwascollapsedbytetrachlorosalicylanilide (1 p~ added a t each point indicated)and completely inhibited by 1 mM NEM. C,polypeptide composition of immunoprecipitated enzyme. Protein was eluted from 300 p1 of beads with pH 2.5 glycine buffer as in the experiments in Fig. 3 and applied to a 12.5% SDS-polyacrylamide gel. Polypeptide composition was identical to that obtained on the immunoaffinity column.
for their ability to stimulate ATPase activity when added tophosphatidylglycerol (PG) addition resulted in stimulation of the immunoprecipitated enzyme. Addition of phosphatidyl NEM-sensitive ATPase activity, as shown in TableI and Fig. ethanolamine,cardiolipin,sphingomyelin,and lysolecithin 5. PG and phosphatidylinositolproduced the greatest stimuproduced eitherinhibition or no effect onNEM-sensitive lation, with up to a &fold stimulation of activity by PG in ATPase activity on the beads (Table I). In contrast, phospha- some experiments. Phosphatidylserineshowed the least stimtidylserine,phosphatidylcholine,phosphatidylinositol,and ulatory activity with a maximum stimulation of less than 2fold. None of the lipids were able to restore activity lost by TABLEI vigorous detergent washing of the enzyme on the bead, alEffect of lipids on NEM-sensitive ATPase activity though the polypeptide composition of enzyme bound to the For each assay, 10 pl of a 10% suspension of staphylococcal beads bead remained unchanged. In subsequent experiments, 100 was coated with rabbit anti-mouse antibody followed by H6.1 antipg/ml PG was added to the ATPase assays. Under these body as described under "Experimental Procedures." The beads were washed once with 25 p1 of T A buffer with 1%polyethylene glycol and conditions, the specific activity of NEM-sensitive ATPase incubated with detergent-solubilized membranes containing 1% pol- activity bound to thebeads varied between 1.2 and 3.1 pmol/ yethylene glycol for l h a t 22 "C. The beads were then centrifuged min/mg. Since the average specific activity of the solubilized and washed once with 1 mlof 150 mM KCl, 30 mM Bis-Tris, 0.1% kidney cortex microsomesfor NEM-sensitive ATPase was CHAPS, 0.1% nonyl glucoside, pH 6.5. The beads were resuspended in 500 pl of 150 mM KCl, 2 mM MgC12,30 mM Bis-Tris, pH 6.5, with 0.015 pmol.min". mg", this represented an 80-206-fold increase in specific activity from the kidney microsomal mem50 pg (100 pg/ml) of lipid as indicated. Assays were performed +1 mM NEM, and each had 6.75 pg of protein and a maximum specific branes, anda 15-20-fold increase in immunoprecipitated speactivity of 1.5 pmol.min". mg". cific activity over values obtainedpreviously without addition Lipid added Activity % of PG (32). The effect of pH on NEM-sensitive ATPase activity of the None 49.5 Asolectin (100) bound enzyme was examined, anda broad optimumat pH6.5 Phosphatidic acid 13.8 was observed (Fig. 6).Subsequentcharacterizationstudies Phosphatidyl ethanolamine 31.0 were done at pH6.5. Cardiolipin 50.2 The nucleotide substrate dependence of the enzyme was Sphingomyelin 94.7 examined (Table 11), and ATP and ITP were the preferred Phosphatidyl serine 122 substrates. GTPase activity in the preparationwas 27% that Phosphatidyl choline 147 Phosphatidyl inositol 245 of ATPase activity. These results are similar to those obtained Phosphatidyl glycerol 278 for the partiallypurified chromaffin granule H'ATPase (17).
15786
Immunoaffinity Purification of Vacuolar H+ATPase 300
-
PC A
..........PI
....................................
a”.
FIG.5. Effect of lipids on ATPase activity. H+ATPase was immunoprecipitated on staphylococcal beads, as described for Table I, except that following incubation with the solubilized membranes, the beads were washed once with 1 mlof 30 mM Bis-Tris-gluconate, 0.1% CHAPS, 0.1% nonyl glucoside, pH 6.5, and resuspended in 500 pl of 30 mM BisTris-gluconate 2 mM MgCls, pH 6.5;lipids sonicated in 10 mM Tris-gluconate pH 7.5 were added to the tubesinconcentrations indicated. Assays contained 4.7 pg of protein with maximum specific activities of 1.48 pmol .min” .mg” protein.
0 %
C
--
-,E 0
U
”””.PC
“ “
A
” ” ”
A
PS
.......... ...........................................................................................
Z
0
1
K
”
I
I
I
1
1
I
I
1
1
1
1
ZW
100
pg lipid/ml
......................
......................
A
A
1.8
‘84
LY?
E
*OI I
.-E
7&
0
n
51)
12
6.0
7.0
8a
30
PH FIG.6. Effect of pH on ATPase activity. H+ATPase immunoprecipitated on staphylococcal beads was assayed as indicated under “Experimental Procedures” a t varying pH values. Assays contained 13 pgof protein with a maximum specific activity of 0.903 pmol. min”.mg”.
E,
.-> .-
.8
w
P .4
TABLE I1 Nucleotide substrate dependence of ATPase activity ATPase affinity purified on monoclonal antibody-coated staphylococcal beads was assayed under “Experimental Procedures”; 100 pg/ml phosphatidyl glycerolwas added to each sample. Samples contained 4.0 pg of protein and had a maximum specific activity of 1.5 pmol .min” .mg” protein. Nucleotide
Activity % ’
ATP ITP UTP GTP CTP
(100)
95.8 44.5 27.4 19.2
The concentration dependence for ATP was examined and found to have a K,,, of 150 p M (Fig. 7). The effect of the anion and cationcomposition of the buffer on NEM-sensitive ATPase activity was examined (Table 111). ATPase activity was not affected by substitution of lithium or potassium for sodium or by complete removal of monovalent cations from the buffer. Divalent cation substitution for magnesium in the assay buffer showed that manganese sup-
S ,
1
.2
1
1
1
.4
1
.6
1
,
.8
1
1
1
1
1
1.0
[ATP] mM
FIG.7. ATP concentration dependence of ATPase activity. H+ATPase immunoprecipitated on staphylococcal beads was assayed as indicated under “Experimental Procedures” in the presence of an ATP-regenerating system. K , was 150 p ~ .
ported full activity, cobalt and calcium partial activity, and barium and strontium little or no activity. 1 mM zinc and copper completely inhibited activity with magnesium present. These findings are in agreement with the properties of the enzyme in thenative vesicles. NEM-sensitive ATPase activity was not affected by substitution of gluconate for chloride in the buffer. The concentration dependence of inhibitors of H’ transport on both bound NEM-sensitive ATPase activity and on proton transport in the unfractionated bovine kidney microsomal
Immunoaffinity Purification of VacuolarH+ATPase
15787
TABLEIV TABLE111 Effect of inhibitors on ATPase activityand proton transport Effect of ionic composition on H’ATPase activity ATPase affinity purified on monoclonal antibody-coated staphyATPase affinity purified on monoclonal antibody-coated staphyFor lococcal beads was assayedas under “Experimental Procedures”;100 lococcal beads was assayed as under “Experimental Procedures.” to each sample. ATPase assays the monovalent ion dependence experiments, assays contained 30 pg/ml phosphatidylglycerol was added contained an average of 8.5 pg of protein and had an average maximM Bis-Tris-gluconate, pH6.5,6mMMg gluconate. The monovalent cation substitutions contained 150 mM of the chloride salt of each mum specific activity of 1.92pmol. min-‘. mg” protein. Proton transion; the monovalent anion experiments contained 150 mM of the port wasmeasuredby acridine orange uptake as describedunder sodium salt of each ion. For the divalent ion dependence experiments, “Experimental Procedures”; samples contained25 Kg of kidney miassays contained 30 m M Bis-Tris-gluconate, pH 6.5, and 2 mM of crosomes. (A) half-maximal concentrations for agents inhibitory to each divalent cation. A, effect of monovalent cations. The chloride bothATPaseactivity and proton transport. (B) effect of agents, salts of the cations indicated were included at 150 mM. Assays had added at concentrations indicated,on bead immunopurified ATPase 4,4‘-diisothiocyanatostilbene-2,2’-disulfonicacid; DIDS, 12.0 pg of protein, with a maximum specific activity of 1.78 pmol. activity. min“ .mg” protein, B, effect of monovalentanions. The sodium salts NBD-CI, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. of the anions indicated were included at 150 mM. Assays had 8.4 pg KIP Inhibitor of protein witha maximum specificactivity of 1.17 pmol .min” .mg“ H+ Transport ATPase activitv protein. C, effect of divalentcationreplacement.Magnesium was replaced by the divalent cations indicated at a concentration of 2 mM; &M PM 100 pg/ml phosphatidyl glycerol was added to each sample. The last A two samples contained 2 mm Mg, and 1 mM, respectively, of Cu and NEM 22.2 28.8 a maximum specific activity Zn. Assays had 12.5 pg of protein and had 14.9 NBD-C1 22.5 of 1.76 pmol. min”.mg” protein. 4.9 DCCD 29.9 Activity % 13.8 DIDS 6.5 Zn 315 170 Monovalent cation Aeent % Activih (100) None Na 78.2 B K 88.5 None (100) Li 96.1 93.0 1 mM vanadate 93.1 5 mM azide Monovalent anion 98.9 1 mM ouabain (100) None
c1
Gluconate Br
86.9 80.7 59.6 56.9
clonal affinity columncould, in principle, contain extrapolypeptides, due to nonspecific binding, or could lose subunits so, through dissociation. However, the polypeptide pattern of the Divalent cation H’ATPase here remained the same under several different 0 None detergent and ionic washing conditions. Enzyme with the (100) Mg same polypeptide retained ATP-dependent proton transport 103.1 Mn and the enzymatic properties of the proton pump in the native 28.7 Ca vesicles. However, additional studies, such as a comparison 19.7 co between the structureof the immunoaffinitypurified enzyme 5.3 Sr 4.0 Ba and the native enzyme in plasma membrane made by high 0 +cu resolution electron microscopic methods, will be needed to 0 +Zn resolve this issue. The immunoaffinity purified kidney H’ATPase retained most of the propertiesof the enzyme in the nativevesicles as vesicles was examined next. Table IV provides a summary of the findings. The Klhfor inhibition of NEM-sensitive ATPase judged by ionic requirements and inhibitor sensitivities. The in the half-maximal effects of activity was very similar to thatfor proton transport activity only major discrepancy was DCCD on proton transport in microsomes and ATPase activfor NEM, NBD-C1, 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, and Zn, but differed for DCCD. ATPase activity ity of the immunoaffinitypurified enzyme; this resultmay be was more sensitive to DCCD than was transport activity, a attributable to partitioning of the hydrophobic agent into microsomal preparation. finding whichmay beattributabletopartitioning of the contaminating membranes in the The purified H’ATPase was capable of ATP-dependent acidhydrophobic agent into membrane lipid not associated with the H+ATPase, thereby lowering the “effective” concentra- ification when reconstituted into liposomes while bound to the affinity beads, conditions in which it is likely that each tion. liposome has only a few proton pumps. On the other hand, proton pumps reconstituted intoliposomes under conditions DISCUSSION where the solubilized enzyme was freein solution rather than Vacuolar H’ATPase was purified from solubilized bovine bound to anantibody-coated bead, displayed ATP-dependent kidney microsomes in a single step by use of a monoclonal potential generation, but not acidification, even in the presa previous paper: H’ATPase was ence of valinomycin.2 The enzyme eluted from the immuimmunoaffinity column. In partially purified from bovine kidneymedulla by conventional noaffinity column, when reconstituted into free-floating liandHPLCchromatographyand was foundtoconsist of posomes, spontaneously aggregated into hexagonal microcrys12,000 t o 70,000 talline arrays identical to arrays of the enzyme seen in the multiple different polypeptides ranging from Mr. In the present paper, an entirely independent method of native membrane.3 These observationssuggest the possibility purifying the enzyme is used. The two methodsof preparation that under conditions where proton pumps are densely packed give a highly similar pattern of polypeptides on SDS gels, in the liposome membrane, valinomycin cannot adequately providingstrong evidence thatthesearesubunits of the collapse the potential generatedby active proton transport. H’ATPase rather than contaminants. Is the observed polypeptide composition correct? An enzyme purified on a monoD. Brown, S. Gluck, andJ. Hartwig (1987)J. Cell Bwl., in press.
15788
Immunoaffinity Purification
The structure of the immunoaffinity purified kidney H+ATPase is similar to thatfor plant (18-20), fungal (21,22), chromaffin granule (17, 23), and coated vesicle (24) H'ATPases, except that no 115-kDa polypeptide was apparent asreported for the lattertwo enzymes (23,24). The kidney proton pump has several enzymatic properties which distinguish it from those reported for other vacuolar H+ATPases. The kidney enzyme is not stimulated by chloride, as reported for the oat root tonoplast H'ATPase (20). The immunoaffinity purified kidney H'ATPase was stimulated greatly by phosphatidyl glycerol and phosphatidyl inositol, and only sightly by phosphatidyl choline and phosphatidyl serine, in contrast to results reported for the chromaffin granule (23) and coated vesicle enzymes (24). Since for both of these enzymes the 115kDa polypeptide has been reported to have ATP binding (23) or ATPaseactivity (25), the presence of this polypeptide may account for the differences in lipid stimulation. The enzyme from kidney does not have this polypeptide but still retains proton transport and ATPaseactivity with specific activities as high as 3.1 pmol. min-' .mg". DCCD-binding polypeptides of M , 12,000-17,000 have been noted for several of the vacuolar enzymes (19,21,23,26). The 15,000 M , polypeptide of the kidney H+ATPase also has a high affinity DCCD-binding site.4 In some of the vacuolar enzymes, it has been demonstrated that the DCCD-binding polypeptide is different from nucleotide-binding sites thought to be the catalytic site (19, 22). These findings suggest that the vacuolar H+ATPase has structural and functional similarities tothe FoFl H+ATPases. Exquisite sensitivity to DCCD is acharacteristic of the FoFl ATPases, in which DCCD binds toan acidic residue in the "DCCD-binding protein," one of the integral membrane polypeptides comprising the transmembrane proton channel. DCCD inhibits the mitochondrial FoF, ATPase with a KIAof 100 nmol/mg protein (27), giving a KIAof 1 pM for 10 pg of protein as used in the kidney inhibition experiments described here. When the catalytic (F,) domain is detatched, the sensitivity of the mitochondrial F1 ATPase to DCCD drops over 10-fold (28), and inhibition occurs by reaction of the reagent with the p subunit (28).The kidney H'ATPase had aK,, for inhibition by DCCD of 4.9 p ~ similar , to the value of 1 pM reported for the oat root enzyme (20), but differing from the values of 130 p~ reported for the coated vesicle enzyme (29). It is possible that differences in the sensitivity to DCCD and differences in other enzymatic properties observed between the kidney and other vacuolar H'ATPases could be the result of detachment of a catalytic portion of the enzyme in a manner analogous to that in the mitochondrial enzyme. Binding of DCCD to polypeptides other than those in the membrane spanning domain might not be apparent at the low concentrations of DCCD generally used with radiolabeled probes. Several properties of the immunoaffinity purified kidney H+ATPase were similar to those of other reported vacuolar H+ATPases. The broad pH profile with an optimum at 6.5 is similar to thatreported for the oat root tonoplast H+ATPase. The substrate specificity of the kidney H+ATPase is similar to that reported for the chromaffin granule enzyme (23), and the K , for ATP is close to that reported for the oat root proton pump (20). The differences in polypeptide composition between medullary and cortex H'ATPase preparations, and theseparation of ATPase activity on the Mono P column into two distinct peaks with differing polypeptide compositions supported the possibility that two or more different forms of the proton pump exist in the affinity purified preparation. This would
' S. Gluck, unpublished material.
of Vacuolar H+ATPase not be entirely unexpected, as the microsomal membranes used to prepare the enzyme are derived from a variety of cell types and likely include a mixture of several different vacuolar compartments. Different types of proton pumps, if present, could therefore represent compartment-specific or cell typespecific forms of the enzyme. The dual pH optimum of ATPase activity found for the affinity purified enzyme could represent the summation of a mixture of enzymes with different pHoptima. The difference in structure between the kidney H'ATPase andthose of other reported mammalian vacuolar-type H+ATPases (17-24) may be due to a difference in the method of preparation or purity of the enzymes or it may reflect an actual difference in structure. The proton pump in mammalian kidney is expressed at high levels and is present predominantly in the plasma membrane in specific cell types in several nephron segments.' The H'ATPase in proton-transporting epithelia also displays rapid changes in compartmentation between intracellular vacuoles and the plasma membrane in response to physiological stimuli (30) not described for proton pumps in othercompartments of the vacuolar system. Proton pumps in different organelles may vary in packing densities, enzymatic properties (31), and may require different targeting information. Having structural differences between proton pumps in different membrane compartments is one mechanism by which the cell could accomplish this. Acknowledgments-We thank Dr. Mary Yurko for assistance with the immunoprecipitation experiments, Dr. Ferenc Kezdy for helpful suggestions, and Donna Kelly for assistance in preparing the manuscript. REFERENCES 1. Forgac, M., Cantley, L., Wiedenmann, B., Altstiel, L., and Branton, D. (1983) Proc. Natl. Acad. Sci. U. A. 80, 1300-1303 2. Glickman, J., Croen, K., Kelly, S., and Al-Awqati, Q . (1983) J. Cell Biol. 97,1303-1308 3. Stone, D., Xie, X-S., and Racker, E. (1983) J. Bwl. Chem. 2 5 8 , 4059-4062 4. Okhuma, S., Moriyama, T., and Takano, T. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2758-2762 5. Yamashiro, D. J., Fluss, S. R., and Maxfield, F. R. (1983) J. Cell Biol. 9 7 , 929-934 6. Van Dyke, R. W., Hornick, C. A., Belcher, J., Scharschmidt, B., and Havel, R. J. (1985) J. Biol. Chem. 2 6 0 , 11021-11026 7. Gluck, S., Kelly, S., and Al-Awqati, Q. (1982) J. Biol. Chem. 2 5 7 , 9230-9233 8. Gluck, S., and Al-Awqati, Q. (1984) J. Clin. Znuest. 7 3 , 17041710,1984 9. Anderson, R. G. W., Falck, J. R., Goldstein, J. L., and Brown, M. S. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,4838-4842 10. Hoogenraad, N., Helman, T., and Hoogenraad, J. (1983) J. Zmmunol. Methods 61,317-320 11. Schneider, C., Newman, R. A., Sutherland, D. R., Asser, U., and Greaves, M. F. (1982) J. Biol. Chem. 2 5 7 , 10766-10769 12. Kagawa, Y., and Racker, E. (1971) J. Biol. Chem. 246, 54775487 13. Loh, Y. P., Tam, W. W. H., and Russell, J. T. (1984) J. Biol. Chem. 259,8238-8245 14. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Bwl. Chem. 193, 265-275 16. Samelson, L. E., Harford, J. B., and Klausner, R. D. (1985) Cell 43,223-231 17. Percy, J., Pryde, J., and Apps, D. (1985) Biochem. J. 231,557564 18. Mandala, S., and Taiz, L. (1985) Plant Physiol. 78, 327-333 19. Manolson, M. F., Rea, P. A., andPoole, R. J. (1985) J. Bwl. Chem. 260,12273-12279 20. Randall, S. K., and Sze, M. (1986) J. Biol. Chem. 2 6 1 , 13641371 D. Brown, S. Hirsch, and S. Gluck, manuscript in preparation.
s.
Immunoaffinity Purification of VacuolarH+ATPase
15789
21. Uchida, E., Ohsumi, Y., and Anraku, Y. (1985)J. Biol. Chem. 28. Pougeois, R., Satre, M., and Vignais, P. (1979)Biochemistry 18, 260,1090-1095 1408-1413 22. Bowman, E. J., Mandala, S., Taiz, L., and Bowman, B. (1986) 29. Sun, S.-Z., Xie, X.-S., and Stone, D.K. (1987)Kidney Zntl. 31, Proc. Natl. Acad. Sci. U.S. A . 83,48-52 183a 23. Cidon, S., and Nelson, N. (1986)J. Biol. Chen. 261, 9222-9227 30. Gluck, S., Cannon, C., and Al-Awqati, Q.(1982)Proc. Natl. Acad. 24. Xie, X-S., and Stone, D.K. (1986)J. Biol. Chen. 261, 2492Sci. U. S. A . 79,4327-4331 2495 31. Mellman, I., Fuchs, R., and Helenius, A. (1986)Annu. Reu. 25. Xie, X-S., Stone, D. K., and Racker, E. (1986)Kidney Znt. 29, Bwchem. 66,663-700 378 32. Yurko, M., and Gluck, S. (1987)J. Bwl. Chem. 262,15770-15779 26. Bowman, E. J. (1983)J. Biol. Chem. 268,15238-15244 27. Linnett, P. E., and Beechey, R. B. (1979)Methods Enzymol. 65, 472-518
