(H+,K+)-ATPase - The Journal of Biological Chemistry

Vol. 261, No. 27, Issue of September 25,, PP . 19060-19065,1992 Printed in U.S. A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Location of the Cytoplasmic Epitopefor a K+-competitiveAntibody of the (H+,K+)-ATPase* (Received for publication, March 26, 1992)

Denis Bayle$, Jean Claude Robert$,Krister BambergQ, Fatima Benkoukall, Anne Marie CheretS, Miguel J. M. Lewin$, George SachsQ, and Annick SoumarmonSII From the Slnstitut National de la Santh et dela Recherche Midicale Uniti 10,H6pital Bichat, 170 Boulevard Ney, 75018 Paris. France. the SCure Institute. Wadsworth Veterans Administration Hospital, Los Angeles, California 90073, and the qFaculte des Sciencesde Rabat, Rabat, Morocco

The monoclonal antibody (mAb) 96-1 11binds thea subunit of (H+,K+)-ATPaseand inhibits the K+-ATPase activity. To map the epitope, all of the partial sequences of the a subunit were expressed Escherichia in coli HBlOl using rabbit a subunit cDNA restriction fragments ligated into PuExvector. Bacterial recombinant lysates were separated by sodium dodecyl sulfate-gel electrophoresis, and the epitope was detected by Western blotting. The antibody site was mapped between Cyss”’ and Glue‘’. This is close to the Lyse’’ that bindsfluoresceinisothiocyanate(FITC) and is considered to be between M4 and M5 close to the ATP binding domain. However, the mAb inhibition of ATPase is not ATP-competitive but is K+-competitive with a KIof 2 X lo-’ M. The mAb also inhibits K+ quench of FITC fluorescence competitively witha Kr of 8 X lo-’ M. The K+ activation of ATPase activity andquench of FITC fluorescence are dependent on K+ binding to an E2 form of the enzyme fromthe extracytoplasmic surface. The mAb epitope is cytoplasmic since the K+ATPase activity of ion-tight gastric vesicles is inhibited. The ‘261-mAb96-111 binds to a single class of sites with an apparentK Dof 2.3 2 0.8 X lo-’ M and K+ does not displace bound mAb. Hence, antibody binding to a cytoplasmicCyssze-G1~661epitopeallosterically at extracytocompetes with K+-dependent reactions plasmic sites.

phorylation site and ATP binding domain. ATP binding has been followed bydetermining the location of ATP-protectable derivatization of cytoplasmic lysines, by either FITC’ or pyridoxal phosphate (8-10). Thus,LYS~’~,Lys618,Lys708, and L Y S ~are ’ ~ protected from reaction by ATP presuming that the ATP-induced conformation of the enzyme prevents exposure of these lysines to thechemicals reagents. Functionally, the (H+,K+)-ATPaseexchanges cytoplasmic H+ for extracytoplasmic K+ as a function of phosphorylation and dephosphorylation at Asp387.Countertransport of these ions is thought to depend on conformational changes in the cytoplasmic domain of the enzyme, transmitted across the membrane domain of the enzyme. It is important therefore to define functional sites on the enzyme to specify where these changes might be occurring and theirrelationship to MgATP and cation binding sites. Potassiumbinds from the extracytoplasmic surface of (H+,K+)-ATPase andinduces conformational changes in the cytoplasmic domain since FITC fluorescence is quenched, trypsinization sites changed, and Asp387dephosphorylation rate increased (11). K+ binding induces the E2 form of the enzyme. Thus it is important to define where K+ binds the extracytoplasmic side of (H+,K+)-ATPase andhow the binding signal connects to changes of the cytoplasmic domain conformation. Functional K+ extracytoplasmic domains of (Na+,K+)-ATPaseshould involve the loop between M1 and M2 since the partially K+-competitive inhibitor,ouabain, binds in that region (12). Several inhibitors have been used to explore the extracytoplasmic domains of (H+,K+)-ATPase. The gastric (H+,K+)-ATPaseis a member of the phospho- A K+-competitive imidazopyridine, SCH28080, has been rylating class of ion-motive ATPases. Similar to the shown to bind in the same region as ouabain, namely the M1(Na+,K+)-ATPase,it is an (Y-P heterodimer, witha large M2 segments and theintervening extracytoplasmic loop (13catalytic (Y subunit and a smaller glycosylated P subunit (1- 15). This class of compound has effects on FITC fluorescence 3). In the case of the hog enzyme, the mRNA encodes for an similar to those of K+, indicating that their binding to the CY subunit of 1034 amino acids, and the N-terminal methionine extracytoplasmic domain also modifies the conformation of is removed during maturation to give a protein of 1033 amino the cytoplasmic catalytic domain (16). Furthermore, another acids (2). The rabbit (Y cDNA encodes for a protein of 1035 K+-competitive antagonist of that class, MDPQ, undergoes amino acids (4). The secondary structure of catalytic subunits fluorescent changes when the enzyme is phosphorylated (17), of this class of ATPases is not defined. The Ca2+-ATPases indicating that the conformation of this extracytoplasmic are thought to have 10 membrane segments; the (Na+,K+)- domain changes reciprocally with cytoplasmic catalytic doATPases, 8 or 9; and the (H+,K+)-ATPase,8 or 10 (1, 4-6). main modifications. The catalytic domain of (H+,K+)-ATPaseshould be located In this work, a functional domain of (H+,K+)-ATPasewas a t least in part between M4 and M5, including the phos- defined using mAb95-111 (18, 19). This antibody inhibits * T h e work was supported by AFLM (Association Franqaise de Lutte contre la Mucoviscidose). The costsof publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “aduertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed INSERM U10, H6pital Bichat, 75018 Paris, France. Tel.: 33-140-25-83-94; Fax: 33146-27-85-36.

Theabbreviationsused are: FITC, fluoresceinisothiocyanate; mAb, monoclonal antibody; EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid; SDS, sodium dodecyl sulfate; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid; pNPP, p-nitrophenyl phosphate;TPCK, L-1-tosylamido-2-phenylethylchloromethyl ketone; MDPQ, l-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-~]quinoline.

19060

K" Domain of the Gastric (H+,K+)-ATPase (H',K+)-ATPase competitively withK+. The epitope was mapped by molecular biology techniques whereby restriction enzymes were used to fragment the cDNA of the a subunit. The fragments were ligated into anEscherichia coli expression vector, and Western blotting of recombinant clones allowed localization of the epitope within a sector of 31 amino acids. It was further shown that the antibody was competitive with K+ activation in ion-tight vesicles when they were treated with nigericin but that K+ did not preventbinding of antibody. EXPERIMENTALPROCEDURES

Materials Diaminobenzidine,agar, phosphoenolpyruvate (monocyclohexylammonium salt), and ATP (M2+ or Na+ salts) were purchased from Sigma. Tris, EGTA, sodium dodecyl sulfate (SDS), chloramine T, and hydrogen peroxide were from Merck (Federal Republic of Germany). Trisacryl2000 was purchased from IBF (France). The restriction enzyme MunI, bovine serum albumin, Hepes, dithiothreitol, were from Boerhinger-France (Meylan, France). The restriction enzymes PstI, PuuII, and HincII, the alkaline phosphatase (calf intestinal), the T4 DNA ligase, and the T7sequencing kit were from Pharmacia LKB Biotechnology Inc. (Les Ulis, France). The yeast extract and the pancreatic hydrolysate of casein were from BioMBrieux (Charbonnibres les Bains, France). The agarose (SeaKem GTG, Nusieve andSeaPlaque) were from FMC(Tebu,LePerray,France).The nitrocellulose filters (0.22 pm) were from Schleicher & Schuell. The [cY-~'S]~CTP and Iz5Iwere from Amersham Corp. the plasmids puEx 1, 2, and 3 were provided by Keith Stanley (Sidney, Australia). The rabbit a subunit cDNA of (H+,K+)-ATPasewas used (4). Methods Epitope Mapping Preparation of Recombinants-The puEx vectors (20) were purified, opened, and dephosphorylatedaccording to Sambrook etal. (21). The rabbit(H+,K+)-ATPase 01 cDNA inBluescript was cutwith restriction enzymes, and the fragmentswere ligated in 10pl containing 100 ng of dephosphorylated puEx vector at an approximative vector/fragment ratio of0.2:1 (mol/mol). The E. coli HBlOl were transformed with the puEx recombinants using a method modified from Hanahan et al. (22). Screening-The transformation medium was spread on LBA agarose Petri plates, and the bacterial clones were grown overnight a t 30 "C. The clones were replicated on nitrocellulose filters, either by overlaying each plate with a nitrocellulose filter or by scarring each clone on a nitrocellulose filter, laying the filter ona freshly prepared Petri plate, and growing the bacteria for several hours a t 30 "C. In both conditions, thefusion proteins were expressed by incubating at 42 "C for 2 h (20). The nitrocellulose filters were then incubated in 5% SDS for 30 min in a 70 "C oven, and the excess SDS was rinsed away in a standard Western blot apparatus (Tris-glycine buffer, 5 volts/cm, 30 min) (23). The nitrocellulose filters were washed for 5 rnin in 40 mM Hepes-Tris, pH 7.0, 0.1% bovine serum albumin, 0.2% Tween 20, 10 mM benzamidine, and 10pg/ml DNase I and then washed for 30 min in the samebuffer without the DNase. Analysis of Recombinants by WesternBlotting-Bacteria were grown in 1 ml of LBA (10 mg/ml Bacto-tryptone, 5 mg/ml Bactoyeastextract, 5mg/ml NaC1, and 100 pg/mlampicillin, pH 7.5) overnight a t 30 "C, diluted 1:25 in LBA, and induced at 42 "C until the A ~ w reached 0.8 unit. A sample (1ml) was centrifuged, the pellet was suspended in 100 pl of 10% sucrose, 1.5 mM EDTA, 100 pg/ml lysozyme, 10 mM benzamidine, and 100 mM Tris, p H 8, at 4 "C. The cells were incubated on ice for 15minand 100 p1, of200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 100 mM Tris, p H 6.8, were added. Asample (10pl corresponding to3 X lo6bacteria/ lane) was loaded on a 10% SDS-polyacrylamide gel. The transfer of proteins on nitrocellulose sheets was performed as described previously (18).The immunological screening was run using 100,000 cpm/ ml "'I-mAb 95-111. Sequencing-The plasmids were purified by NaOH lysis of bacteria according to Qiagen (Qiagen, COGER, France). The T7 sequencing kit was used as indicated by Pharmacia.

19061

Hog Gastric Fractions Fresh hog stomachs were obtained from a local slaughterhouse (Domont Abattoirs, Pontoise) and treated as describedpreviously (24). All steps were at 4 "C. The homogenate was centrifuged for 10 min a t 800 X g, and the resulting supernatant was centrifuged at 40,000 X g for 7min (SorvallRC5C). The supernatantwas centrifuged a t 100,000 X g for 30 min (Beckman L5 65). The microsomal pellet was either suspended in 42% sucrose and equilibrated by running under a 30-35% stepped sucrose gradient (ion-permeable vesicles) or suspended in 0.25 M sucrose and equilibrated on a 7% Ficoll, 0.25 M sucrose cushion (ion-tightvesicles). Proteins Proteins were measured by the method of Bradford, using bovine serum albumin as standard (25). (H+,K+)-ATPaseActivity The activity was measured a t 37 "C by adding 5 pg of protein to 1.2 ml of 40 mM Hepes, pH 7.0, 2 mM MgATP, 5 p M nigericin, 4 mM phosphoenolpyruvate, 1 unit/ml pyruvate kinase, with or without 20 mM KC1. pNPPase Activity The activity was measured a t 37 "C by adding 5 pg of protein to 250 p1 of 40 mM Hepes-Tris, pH 6.9, 2 mM pNPP, 2 mM MgCI, with and without 20 mM KC1. Monoclonal Antibody 95-1 11 mAb 95-111 was as described previously (18, 19). mAb 95-111 was purified by DEAE-Affi-Gel blue chromatography (26). Iodination of mAb 95-1 11 Iodination was performed essentially as described by McConahey (27). Aliquots of mAb 95-111 (50 pl, 1 mg/ml) were diluted in 50 pl of 0.1 M phosphate buffer, pH 7.5, and 1 mCi of "'I (10 pl)was added followed by chloramine T (1 pg in 5 p1 of phosphate buffer). After 10 min, the reactionwas stopped by adding freshly prepared sodium metabisulfite (5 pg in 10 p1of phosphate buffer). Free radioiodine was separated from '"I-IgG by chromatography on Trisacryl2000(20 X 1-cm column) equilibrated with 0.1 M phosphate buffer plus 0.1% bovine serum albumin. The first radioactive peak elutedcorresponded to the radiolabeled mAb 95-111 IgG, and lo6 cpm corresponded to 1.3 X lo-" mol. Epitope Titration Hog gastric membranes were diluted to 80 pg of protein/ml in 15 mM carbonate, 35 mM bicarbonate, and100-p1aliquots were incubated overnight a t 0-4 "C in the wells of polyvinyl chloride microplates (Flow Laboratory). The solutionswere decanted and thewells rinsed (4 X 5 min) with 200 pl of 50 mM Hepes-Tris, pH 7.2, 0.1% bovine serum albumin (buffer A).The lastrinse was left for 1 h. Radiolabeled monoclonal antibody (100 pl, 7.5-500 ng) was diluted in buffer A and incubated for 2 h in the coatedwells at room temperature. The plates were rinsed four times with buffer A, cut out, and theirradioactivity counted (1275 minigamma LKB). FITC Fluorescence Hog gastric membranes (100 pg) were treated for 120 min with 5 freshly prepared FITC and rinsed as described by Jackson et al. (10). The FITC-treated pellet was suspended in 0.25 M sucrose, 20 mM Tris, pH 7.4. The fluorescence was measured with excitation a t 495 nmand emission a t 517 nm using theLS-5BPerkin-Elmer spectrofluorometer.

pM

Trypsinization Hog gastric membranes (1.5 mg of protein/ml) were digested at 37 "C with TPCK-treated trypsin (33pg/ml) in 50 mM Tris-HC1, pH 6.8, for 5-40 min. The proteolysis was stopped with 10 pg/ml N"-ptosyl-L-lysine chloromethylketone. Two SDS-polyacrylamide gel electrophoreses were run in parallel; one was stained with Coomassie Blue, and the other was transferred on nitrocellulose and incubated with "'I-mAb 95-111 (lo6cpm/ml) for Western blotting.

K' Domain of the Gastric (H+,K+)-ATPase

19062 1 2 345

(H+,K+)-ATP~s~a CDNA

'

5' -69 1

RESTRICTION ENZ. \

.

E

Pstl 3

FIG.1. Western blot oftrypsinized(H+,K+)-ATPase.

Hog gastric membraneswere trypsinized in the presence of 25 p~ Schering 28080,0.22 mM ATP, 1 mM EDTA, 2 mM MgC12, 1.5 mg of protein/ ml, and 2% (w/v) trypsin at 37 'C. Trypsinization induces the rapid accumulation of two wide bands a t 64 and 34 kDa. Only the 64-kDa band is immunoreactive. The left part of the figure shows the gel stained with Coomassie Blue (lanes 1 3 ) ; the right part (lanes 4 and .5), the autoradiography obtained after an incubation with 10' cpm/ ml ""I-mAb 95-111. Lane 1, standards (95, 67, 60, 43, 36, and 30 kDa); lanes 2 and 4, no trypsinization (2.5 pg of protein); lanes 3 and 5 , 40-min trypsinization (6pg of protein).

' 3457

PStl

-

LIGATED FRAGMENTS

1589

PStI HincII

-

81 1

HincII

3'

3105

HincII HincII + PvuII 811

HincII + PvuII

-

2730

3457

I 998 .-

2569

3457

1500 1501

HincII + PvuII + MvnI

2729

1859

rn 1572 1686

FIG.3. mAb 95-1 11 epitope mapping. The cDNA of rabbit N (H',K')-ATPase (top of the figure) was fragmented using the restriction enzymes listed on the left of the figure. The 811-1998 fragment obtained with HincII waspurified by agaroseelectrophoresis and restricted with PuuII. The 1501-1859 fragment obtained with HincII and PuuII was purified and restrictedwith MunI. The fragmentswere ligated in puEx, and plasmidswere sequenced to assure thatonly one cDNA fragment was ligated. After expression of proteins, the clones were tested for reactivity with mAb 95-111 by dot and Western blots: m, immunoreactiveclones; , nonimmunoreactive clones. HK Rabbit 529 CSSILIKGQE LPLDEQWREA FQTAYLSLGG LGE 528 CSSILIKGQE LPLDEQWREA FQTAYLSLGG LGE HK Hog

FIG.2. Characterization of mAb 95-1 11epitope by expression of (H+,K+)-ATPase cDNA restrictionfragments. The (H',K+)-ATPase a cDNA was fragmented and ligated to the puEx plasmids; transformed bacteria were grown and fusion proteins expressed as detailed under "Methods." The bacteria werepelleted, lysed in SDS,and loaded on10% polyacrylamidegels (3 X lo6 bacteria/lane). The right part of the figure shows the proteic pattern, stained withCoomassie Blue; the leftpart, theautoradiographic pattern after transferof proteins onto nitrocellulose for Western blot with '"I-mAb 95-111 (10' cpm/ml for 2 h at room temperature). The ATPase cDNA insert present in loaded material is identified on top of each lane by the numbers of starting and endingnucleotides. PuEx control is the plasmid with no insert. Standards are 220, 95, 67, and 60 kDa. The arrow indicatesthe @-galactosidaseencoded by the control plasmid puI3x. RESULTS

Mapping by Trypsinization-After the proteolysis of the E2-P (H',K')-ATPase with 2% trypsin, the epitope was prese n t in a 64-kDa fragment (Fig. 1) (amino acid positions 49672) which, as reported previously by Munson et al. (15),was accumulated rapidly and then hydrolyzed slowly. The complementary peptide accumulated around 34 kDa but was not immunoreactive. This suggested that the epitope is mapped between the amino acids G ~ and u Argfi". ~ ~ In the presenceof K', the immunoreactive trypsinized fragment was the 56-kDa fragment, indicating that the epitope should be between Ile4" and Argfi72(28). Epitope Mapping by cDNA Restriction and ExpressionThe cDNA of rabbit a (H',K')-ATPase subunit was fragmented with PstI or HincII, and the large fragments were ligated in puEx after the P-galactosidase sequence. The epitope-expressing clones were selected by dot blots anddouble checked by Western blots. The bacteria infectedwith the control puEx plasmid expressed a nonimmunoreactive 120kDa @-galactosidase(Fig. 2). The other clonesexpressed different sizes of fusion proteins, and some were immunoreac-

HK R a t HK Man

528 CSSILIKGQE LPLDEQWREA FQTAYLSLGG LGE 529 CSSILIKGQE LPLDEQWREA FQTAYLSLGG LGE

100% 100% 100% 100%

FIG.4. mAb 95-111 epitope. Primary sequences ofmAb95111 epitopes in rabbit (4), hog (2), rat ( I ) , and man (31) (H*,K+)ATPase (HK)and the corresponding sequences in nl, a2, and 03 chains of (Na',K')-ATPases (NKase) ( 3 2 ) and in sarcoplasmic reticulum Ca"-ATPase (Case) (33). Percentages of homology are given underlined, on theright of the figure. Nonhomologous amino acids are and carboxylic amino acids are boldface. The most probable secondary structure of the sequence is an amphipatic n helix. tive. The plasmids of immunoreactive clones were sequenced to discard thosewith several cDNAfragments andcheck that reading framewas correct. The first enzyme, PstI, restricted the a cDNA into three fragments: 336-1588, 1589-2729, and 2730-3457. The immunoreactive sequence was encoded by the 1589-2729 fragment only (Fig. 3). HincII restricted a cDNA into four fragments: (-69) to 810,811-1998,1999-2568, and 2569-3457. The 8111999 fragment was the only one encoding for the epitope, indicating that the epitope was mapped between bases 1589 and 1998 (Fig. 3). The 811-1998 HincII fragment was purified and further restricted by PuuII. The smallest cDNA fragment encoding for the epitope containedbases 1501-1859 (Fig. 3). That fragment was purified and restricted with MunI. The only immunoreactive sequence was mapped between bases 1572 and 1686. That fragment encoded for 39 amino acids between Arg?'" and Glu"' in hog (H',K')-ATPase. Part of the sequence was lacking in the immunoreactive 1589-2730 PstI fragment, suggesting that the epitope was finally encoded between the bases 1589 and 1686 sequence and corresponded to the peptidic sequence Cys"g-G1~"61. The epitope is 100% homologous in hog, rabbit, rat, and

K" Domain of the Gastric (H+,K+)-ATPase

19063

either in termsof saturation or affinity. man in agreement with the immunoreactivity of all these The finding that mAb 95-111 was K'-competitive with species (Fig. 4). The equivalent sequences in the (Na+,K+)vesicles ATPase 011, 012, and 013 have 70, 67, and 70% homology, respect to ATPase activity in intact, nigericin-treated respectively, and the homology withthe reticulum sarco- indicates that the antibody binds to the cytoplasmic surface of the ATPase. As K' could not displace bound mAb, the plasmic Ca2+-ATPaseis only 18%. Antibody Binding to Membranous Hog (H+,K+)-ATPme- mAb effects were related to conformations induced by the T h e binding of lZ5I-antibodyto the (H+,K+)-ATPase was antibody elsewhere than inregions of the epitope. The finding saturable with only a single class of sites with an apparent that the antibody was K+-competitive for induction of the E2-FITCconformation suggests that mAb 95-111 atthe Kr, of 2.3 +- 0.8 X lo-' M ( n = 6) (Fig. 5 andTableI). Formation of the FITC derivative did not affect antibody cytoplasmic surface competes with K+ binding to the extracytoplasmic E2 site. affinity (Table I). Interaction of ATP with Antibody-At low concentrations Interaction of K' with Antibody-mAb 95-111 was competitive with K' activation of (H',K')-ATPase using ion-tight of ATP (0.1 mM) in the rangeof the K, for ATP, essentially of "'I-antibody binding. At and ion-permeable vesicles. The V,,, for K+-stimulated ATP no effect was foundinterms hydrolysis remained unchanged in the presence of the mAb, higher concentrations (over 2 mM) an effect was noted but the apparent K, for potassium increased from 3.3 to 20 whereby the affinityof the antibodydecreased (Fig. 7). In the mM in the presence of 6 X lo-' M mAb 95-111 (Fig. 6). The same range of concentrations, neither ADP nor G T P was as effect was specific mAb Kr was 2 X lo-' M using ion-tight vesicles and nigericin effective as ATP(Fig. 8),indicating that the (Table I). The mAb was also a competitive antagonist of K+ for ATP; however, it was presumably nonspecific of the catwith respect to the pNPPase activity, but the mAb KI was alytic ATP sites. In the ATPaseassay using 20 mM KC1, mAb 95-111 was a only 8 X lo-' M (Table I). K' quenches the fluorescence of FITC enzyme by inducing noncompetitive inhibitor with respect to ATP. Thus, in the an E2 conformation. Antibody also quenched FITC fluores- presence of 3 X lo-' M antibody, the K , for ATP was uncence with an apparent KO of x lo-' M. The presence of the changed at 125 pM, whereas the V,,, decreased from 149 to 80 pmol of ATP hydrolyzed per mg/h (Fig. 9). Asimilar antibody did not change the maximal K+-induced quench but increased the apparentK, for K' from 3 to 10 mM, indicating situation was observed for pNPP, in which K, remained at 2 a Kr for antibody of 8 X lo-' M. Hence mAb 95-111 is also mM and the rate decreasedfrom 45 to 30 Fmol of pPNP hydrolyzed per mg/h. K+-competitive in terms of E2-K formation for the FITC enzyme. The presence of K+, with andwithout valinomycin or DISCUSSION MgATP, did not affect lZ5I-antibody binding to the enzyme, Various techniques have been developed for defining epitopes of antibodies.One technique uses an enzyme-linked immunosorbent assay onimmobilized sequential octapeptides and has beenused, for example, to define the epitope for (H+,K+)-ATPase antibodiesHK12-18 (29) and146.' Another approach is theuse of proteolytic digests followed by separation of peptides andsequencing of bands that are both positive and negative on Western blots. Thiswas used in part here to show that mAb 95-111 recognized an epitopein the Nterminal two-thirds of the (Y subunit of the (H+,K+)-ATPase, =lo the fragment obtained withlimited digestion which is known to extend from amino acid 47 to 670. In the presence of K+ a fragment of 56 kDa is produced which begins at Ile45fiand is also recognized by mAb 95-111. Hence, tryptic mappingshows that the epitope is between positions 456 and 670. A powerful mapping approach is to use expression cloning 0 100 500 of cDNA restriction fragments in vectors such as the puEx 95-111 TOTAL (ng/puits) used here. With this method, itwas possible to show that the FIG. 5. Steady-state bindingof mAb 95-111 to hog gastric epitope for mAb 95-111 is located just distal to the FITC membranes. Hog gastric membranes were coated on plastic wells binding site, between Cys"' and G l ~ ' ~ l . and incubated a t room temperature for 2 h with lz5I-mAb 95-111, Another monoclonal antibody which also inhibits ATPase

'i

(7.5-500 ng/100 pl). After severalrinsings, wells were cut and counted. I n the inset, data were plotted in B/F = f ( B ) ,where B is bound and F i s free mAb 95-111.

T. Joys and F. Mercier, personal communication.

TABLE I mAb 95- 111 affinity parameters The affinity of mAb 95-111 was measured by titrating bound FITC-quenching, '251-mAb 95-111 binding to control and FITC-treated membranes. The mAb K, was calculated from the inhibitionof ATPase activities and FITC quenching as described in Fig. 7. Titration mAb K,,, Competition mAb K, 10-9 M

Of mAb effect on FITC quenching Of mAb binding to control membranes Of mAb binding to FITC-treated membranes

With ATP 10 (ATPase) (noncompetitive) 2 With PNPP (pNPPase) (noncompetitive) (competitive) (ATPase) 3 With K With K (pNPPase) (competitive) With K (FITC quenching) (competitive)

lo-' M

2 80 8

K+ Domainof the Garstric (H+,K+)-ATPase

19064 SLOPE

/*

003,

YATP [mM]" 7

0

io 20 [mAb 951111 10-gM

FIG.6. (H+,K+)-ATPase activity: competition between mAb 95-111 and K+. In the inset, (H+,K+)-ATPaseactivity of gastric membranes was measured in the absence ( 0 )and in the presence of mAb 95-111 (A,6.6 x lo-' M ) , (W, 13.2 x IO-' M), (+, 20 x lo-' M ) and the presence of 2 mM ATP and 2 mMMgC1,. In the main figure, slopes of Lineweaver and Burk linear regressions are plotted as a function ofmAh 95-111 concentration in the assay. The mAb K,, extrapolated for y = 0, is 8 X lo-' M in this experiment. Using ionM nigericin to allow K+ activation of the tight vesicles and 5 X ATPase via intravesicular K+ site, the sameinhibitions were observed, and a mAb K, of 2 X lo-' M was obtained.

0

100 95-111 TOTAL

(ng/puits)

FIG.7. Effect of ATP on mAb 95-1 binding 11 to hog gastric membranes. Hog gastric membranes were coated on plastic wells and incubated for 2 h at room temperature with varying concentrations of "'I-mAb 95-111, in the absence (0)and presence of ATP, 6 mM (0)and 20 mM (0).In the inset, data were plotted as B/F = f ( B ) , where B is bound and F is free antibody. In the presence of ATP, the Scatchard plot was biphasic, indicating two different epitopes. The addition of ATP did not change the BmaX.

0

50

loo

m

200

mAb 95111 (ng)

FIG.8. Effect of ATP, GTP, and ADP on mAb 95-111 binding to hog gastric membranes. Hog gastric membranes were coated on plastic wells and incubated for 2 h at room temperature with varying concentrations of IZ5I-mAb95-111 in the absence of and in the presence of 20 mM ADP (01, 20 mM GTP nucleotide (0) (O), and 20 mM ATP (A). The amount of bound antibody is plotted a s a function of the amount of mAb 95-111 incubated per well.

FIG.9. (H+,K+)-ATPase activity:competition between mAb 95-111 and ATP. (H+,K+)-ATPaseactivity was measured using 2 mM MgC1, plus or minus 20 mMKC1, in the presence (0)and in the absence (A)of lo-* M mAb 95-111. The K+-stimulated activity is plotted as a function of ATP concentrations; mAb 95-111 decreased ATPase V,, but had no effect on the affinity for ATP. activity has been mapped previously by proteolysis between amino acid positions 456 and 620 (28, 30). Because of its inactivation after cleavage of ATPase fragments with formic acid, the epitope is probably close to Asp507-Pro508 and Asp5loPro511 and was suggested to include the 504-512 sequence. Both mAb 95-111 and 5B6 are noncompetitive with respect to ATP,although ATP prevents FITC binding to Lys517which is placed between the epitopes in the linear sequence of the enzyme. ATP does not decrease mAb 95-111 binding, nor is it protective in terms of enzyme kinetics. The effect of high concentrations of ATP on mAb 95-111 binding is out of range of the kinetic sites for ATPase activity; therefore, even if ATP effects are specific with respect to ADP and GTP, they must be considered as nonspecific of catalytic sites. Hence, the region of the enzyme between positions 512 and 529 is affected by catalytic ATP to the extent that Lys517can no longer bind FITC, whereas the regions on either side bind antibodies without effect on ATP binding affinity. Although mAb95-111 and 5B6 were similar in terms of interaction with ATP, in the kinetic analysis of ATPase activities, they differed with respect to K+ activation, 5B6 being uncompetitive (30) and 95-111 being competitive. It therefore appears that theconformation induced by antibody 95-111 binding tothe 529-561region is exclusive ofK' binding to its site and that thebinding of an antibody to the 504-512 region inhibits ATPase activity without affecting K+ interaction. Antibody 95-111 is able to precipitate intact inside-out vesicles (24) and is able to inhibit K'-ATPase of intact vesicles that require nigericin for activation. It is clear that the epitope is therefore cytoplasmic. The epitope is located between M4 and M5, thought to be a large cytoplasmic loop in the enzyme, based on hydropathy and chemical modifications. Since the El-Kform is inhibitory and only the E2P-Kform activates the enzyme, competition between antibody and K' relates to the E2P-Kform of the enzyme. Hence competition is between the conformational effect of cytoplasmic binding of the antibody and thebinding of K' to theextracytoplasmic facing conformation of the K+ binding site. Since the cytoplasmic binding of mAb 95-111 restricts K+induced FITC quenching and thus the induction of cytoplasmic conformations by extracytoplasmic K, conformational changes are transmitted across the membrane domain of the enzyme from the cytoplasmic face to the extracytoplasmic domain. Previous results have led to similar conclusions. It is known that K+-competitive inhibitors bind to the

K" Domain of the Gastric (H",K+)-ATPase

19065

8. Tamura. S.. Taeava. M.. Maeda. . . M.. . .and Futai. M. (1989) J. Biol. Chem. loop between M1 and M2 and that a conformational change 264,8580-8&34 9. Farley, R. A., and Faller, L. (1985) J. Biol. Chem. 260,3899-3901 is induced in that region with phosphorylation of the enzyme R. J., Mendlein, J., and Sachs, G. (1983) Biochim. Biophys. Acta at Asp386,suggestingamorehydrophobiclocationfor the 10. Jackson, 781.9-1 5 .- -,- - binding site of the arylquinoline, MDPQ (17). This implies 11. Rabon, E. C., and Reuben, M. A. (1990) Annu. Reu. Physiol. 52,321-344 12. Price, E. M., and Lingrel, J. B. (1998) Biochemistry 27,8400-8408 motion in the extracytoplasmic region induced by phosphoryl- 13. Munson, K. B., and Sachs, G. (1988) Biochemistry 2 7 , 3932-3938 ation in the samecytoplasmic loop containing the epitopefor 14. Keeling, D. J., Taylor, A. G., and Schudt, C. (1989) J. Biol. Chen. 2 6 4 , 5545-5551 antibody 95-111. mAb 95-111 inhibits the phosphorylation 15. Munson. K. B.. Guttierez. C.. Balaii. V. N.. Ramnaravan. " . K..,and Sachs. steady-state level (18),and it may be that the binding of the G. (1991) J. Biol. Chem.'266, 18676-18988 16. Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson, R., Mendlein, u ~ the ~ ~ antibody in theregion between Cys529and G ~ prevents J.. Rabon. E.. and Sachs. G. (1987) J. Biol. Chem. 262. 2077-2084 stabilization of K' binding at Ml"2 by inhibiting the phos- 17. Rabon, E., Sachs, G., Bassilian, S., Leach, C., and Keeling, D. (1991) J. Biol. Chem. 2 6 6 . 12395-12401 phorylation and the induction of a K conformation effective 18. Benkouka, F., Pkranzi, G., Robert, J. C., Lewin, M. J. M., and Soumarmon, A. (1989) Biochim. Biophys. Acta 9 8 7 , 205-211 in destabilizing E2-P. In contrast, binding of antibody 5B6 in 19. Robert, J. C., Benkouka, F., Bayle, D., Hervatin, F., and Soumarmon, A. a region close to the 95-111 epitope in the linearsequence of (1990) Biochim. Biophys. Acta 1024,167-172 the ATPase is not K+-competitive but equally is noncompet- 20. Bressan, M. G., and Stanley, K. K. (1987) Nucleic Acids Res. 1 5 , 1005610064 itive with respect to ATP. Thus regions that appear close in 21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual, 2nd ed, pp. 1.60-1.61, Cold Spring Harbor Laboraterms of linear sequence have quite different structural intertory, Cold Spring Harbor, NY actions with the rest of the protein. 22. Hanahan, D., Jessee, J., andBloom, F. (1991) Methods Enzymol. 2 0 4 , 63'

Acknowledgments-We acknowledge Richard Benarous, Keith Stanley, and George Banting for help with epitope mapping technologies. We also acknowledge Philippe Champeil, Marc Lemaire, and their collaborators for interesting discussions of the work. REFERENCES 1. Shull, G. E., and Lingrel, J. B. (1986) J. Biol. Chem. 2 6 1 , 16788-16791 2. Maeda, M., Ishizaki, J.,andFutai, M. (1988) Biochem. Biophys. Res. Commun. 157,203-209 3. Reuben. M. A,. Lasater. L. S.. and Sachs. G. (1990) Proc. Natl. Acad. Sci. U. S.A. 87,'6767-6711 ' 4. Bamberg, K., Mercier, F., Reuben, M. A., Kobayashi, Y., Munson, K., and Sachs G. (1992) Biochim. Biophys. Acta 1131,69-77 5. Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H. (1986) Cell 44,597-607 6. Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Nature 3 1 6 , 691-694 7. Deleted in proof

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