May 25, 2015 - 263,3652-3656). The ... has a molecular mass of about 100 kDa (2,3) and is classified ... of protein/ml of 50 mM MOPS-NaOH, pH 8.0) were incubated with ... centrifugation (100,000 X g, 1 h) after addition of 100 mM sucrose.
THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 264, No. 15,Issue of May 25, pp. 8580-8584. 1989 Printed in US. A.
0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Pig Gastric (H’
+ K’)-ATPase
LYS-497 CONSERVED IN CATION TRANSPORTINGATPasesIS PYRIDOXAL 5’-PHOSPHATE*
MODIFIED WITH
(Received for publication, December 5, 1988)
Shigehiko Tamura, MitsuoTagaya, Masatomo Maeda,and Masamitsu Futai From the Department of Organic Chemistry and Biochemistry, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567, Japan
Pig gastric (H’ + K+)-ATPasecan be covalently modified with pyridoxal 5’-phosphate (PLP) (about 1 mol/ mol enzyme), and this modification is not observed in the presence of ATP, suggesting that PLP binds to a specific Lys residue in the ATP binding site or the region in itsvicinity (Maeda, M., Tagaya, M., and Futai, M. (1988) J.Biol. Chem. 263,3652-3656). The peptides labeled with radioactive PLP couldbe released from the gastric membrane vesicles quantitatively by chymotrypsin treatment, and two peptides were purified by high performance liquid chromatographies. These peptides were not obtained from vesicles incubated with PLP in the presence of ATP. The sequences of the two peptides were NHz-Asn-Ser-ThrAsn-Lys-Phe-COOHand NHz-Ser-Thr-Asn-Lys-PheCOOH, exactly correspondingto residues 493-498 and 494-498, respectively, of pig gastric (H+ + K+)ATPase sequenced recently (Maeda, M., Ishizaki, J., and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157,203-209). Lys-497 was concluded to be the binding site of PLP, as pyridoxyl-Lys was identified at the corresponding position. This Lys residue is conserved in (Na+ K+)- and Ca2+-ATPases.The possible amino acid residues in the catalytic site of gastric (H+ + K+)-ATPaseare discussed.
+
tion site (8) of the pig enzyme have been determined chemically. But except for the Lys residue that binds FITC and the phosphorylated Asp residue, the essential residues for catalysis by the enzyme have not been characterized. The results of chemical modification experiments suggest that amino (9, lo), sulfhydryl (2,9, 11-13), guanidino (10, 14), and carboxyl (2, 15) groups participate in the catalytic process. However, their locations in thepolypeptide chain have not been determined, as the specificities of the reagents used were low and the stoichiometries of the binding have not been established. Recently, two kinds of reagents were reported to modify (H+ K+)-ATPase specifically with defined stoichiometry. Omeprazole (a substituted benzimidazole) modifies sulfhydryl groups of the enzyme (16), andpyridoxal 5”phosphate (PLP) possibly modifies its t-amino group of Lys residue (3). We suggested previouslythat PLPbinds to a specific Lys residue in the nucleotide binding site or the region in its vicinity (3). In thisstudy, we purified PLP-binding peptides released from gastric membranes by treatment with chymotrypsin and determined their amino acid sequences. The sequence around the Lys residue that binds PLP was found in the primary structure deduced from the cDNA sequence (17).
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MATERIALS AND METHODS
Purification of Gastric (H+ + K+)-ATPase-Membrane vesicles enriched in (H+ + K+)-ATPase were purified from gastric mucosa of Gastric (H+ K+)-ATPase is an intrinsic membrane pro- freshly slaughtered hogs (3). (H++ K+)-ATPase (K+-dependentATP hydrolysis) was assayed a t 37 “C in 20 mM PIPES-triethanolamine tein localized in plasma membranes of the secretory surface (pH 6.8) as the difference between the amounts of inorganic phosof parietal cells and responsible for acid secretion (1).The phate released in the presence and absence of 10 mM KC1 (3). Protein enzyme catalyzes electroneutral exchange of internal H’ and was determined as described (18). Modification of (H+ + K+)-ATPase with PHIPLP-Vesicles (1 mg external K+ coupled with ATP hydrolysis (2). The enzyme has a molecular mass of about 100 kDa (2,3) and isclassified of protein/ml of 50 mM MOPS-NaOH, pH 8.0) were incubated with as an E1E2-type (or P-type) ion-transporting ATPase, as a 30 b M of [3H]PLP in the presence or absence of 2 mM ATP for 20 min at 25 “C (3). After treatment with NaBHl and 2-fold dilution phosphorylated intermediate is formed during its catalytic with 0.1 M Tris-HCl buffer (pH 7.4), the vesicles were collected by cycle (4,5). centrifugation (100,000 X g, 1h). They were suspended in 0.1 M TrisA large amount of membrane vesicles enriched in this HCl (pH 7.4) containing 0.03% (w/v) SDS at a final protein concenenzyme can easily be prepared from pig gastric mucosa (2, 3). tration of 0.6 mg/ml, incubated for 15 min a t 25 “C, andthen The sequences of the 16 amino-terminal residues (6) and the precipitated by centrifugation. Incorporated radioactivity was measregions around the FITC’-binding site (7) and phosphoryla- ured in an Aloka LSC-700 liquid scintillation counter (3). Samples were analyzed by electrophoresis in polyacrylamide gel (10%) con* This research was supported in part by grants from the Ministry taining SDS (19) and thenautoradiography. Radioactivity associated of Education, Science, and Culture of Japan, the Science and Tech- with (H+ + K+)-ATPasewas measured as described previously (3). Modification of (H+ + K+)-ATPase with FZTC-Vesicles (4 mg of nology Agency of the Japanese Government, and the Foundation for the Promotion of Pharmaceutical Sciences. The costs of publication protein in 4ml of 50 mM MOPS-NaOH, pH 8.0) were incubated with of this article were defrayed in part by the payment of page charges. or without 10 p M FITC (dimethyl sulfoxide solution) for 1 h at 25 ‘C. This article must therefore be hereby marked “advertisement” in Then the incubation mixture was applied to a Sephadex G-50 (super fine) column (1.5 x 5.5 cm) equilibrated with 50 mM Tris-HC1 (pH accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The abbreviations used are: FITC, fluorescein isothiocyanate; 8.0). Modified vesicles eluted in the void volume were collected by HPLC, high-performance liquid chromatography; MOPS, 3-(N-mor- centrifugation (100,000 X g, 1 h) after addition of 100 mM sucrose. pho1ino)propanesulfonicacid; PIPES, piperazine-N,N’-bis(2-ethane- This treatment with FITC caused 90% inhibition of (H+ + K+)sulfonic acid); PLP, pyridoxal 5”phosphate; [3H]PLP, 4-[f0rmyl-~H] ATPase. The vesicles were further incubated with [3H]PLP as depyridoxal 5’-phosphate; SDS, sodium dodecyl sulfate; TLCK, N-a- scribed above. K+)-ATPase with Chymotrypsin-VesiPartial Digestion of (H’ tosyl-L-lysylchloromethyl ketone.
+
+
8580
Lys-497 of Gastric (H'
+ K")-ATPase
85s1
cles were modified with ['HIPLP as described ahove, except that SDSresults suggest that PLP bindsspecifically to the ATP-bindtreatment was omitted. The modified vesicles were suspended in 5 ing site or the region in its vicinity (3). Furthermore, after mM PIPES-triethanolamine (pH 6.8) at a protein concentration of modificationwith ["HIPLP,the enzyme yielded a57-kDa 400 pg/ml andincuhatedwithvariousconcentrations of TLCKfragment on treatmentwith chymotrypsin, and this fragment Chymotrypsin(N-n-tosyl-L-lysvlchloromethylketone-treatedchymotrypsin) on ice for 1.5 min. Proteolysis was terminated hy adding was digested to 17-kDa polypeptides with the intermediate 80 mM Ij-mercaptoethanol. Samples were analyzed by electrophoresis appearance of a 42-kDa polypeptide (Fig. 2), indicating that in polyacrylamide gel (12%) containing SDS. P L P bound to a specific region of the enzyme. Proteolvsis for Isolation of Labeled Peptides-Vesicles (20 mg of Release of Radioactive Peptidefs) by Proteolytic Digestionprotein)were modified with['HIPLPandtreatedwithSDS as On treatment with trypsin, a rather hydrophilic radioactive described ahove. They were then suspended in 5 mM MOPS-NaOH (pH 8.0) and5 mM CaCI? at a protein concentration of 1 mg/ml and peptide is released from the enzyme modified with ["HIPLP incuhated for 4 h at 37 "C in the presence of 5 pg/ml of TLCK- (3), whereas FITC binds to a Lys residue in a hydrophobic chymotrypsin. The reaction mixture was centrifuged (100,000 X g, 1 region (7). Thesefindings suggest that PLP bindsto a differh), and the supernatant was lyophilized. The lyophilizedpeptides ent Lys residue from that modified by FITC. We next examwere dissolved in 5 ml of H20. ined suitable conditions for proteolysis of gastric membrane Purification of Peptides b.y HPLC-The buffers used for HPLC were: solution A, 0.15 trifluoroacetic acid in water; solution R, 0.1% vesicles modified with ["HIPLP tosolubilize radioactive peppepsin, or V8 protease. trifluoroacetic acid and 90% acetonitrile in water; solution c, 10 mM tides with chymotrypsin, thermolysin, ammonium acetate (pH 6.0); solution D, 10 mM ammonium acetate Thermolysin produced a small radioactive peptide(s) which (pH 6.0) and 50% acetonitrile. Chromatography was carried out a t was notretainedonthe reversed phase column used for room temperature using a Shimadzu Gradient-LC6A System (Kyoto, HPLC. Under acidic conditions, the membranes aggregated Japan) and CIRreversedphasecolumns of Shim-packCLC-ODS partial digestion. V8 protease also (Shimadzu) and Cosmosil 5C-18 (Nakarai Chemical Co., Kyoto, Ja- and pepsin caused only released only a small amount of radioactive peptides. Chypan). Amino Acid Sequencinfi-Sequencing was performed in an Applied motrypsin was the best of the proteases examined, causing Riosystems 477A gas-phase sequencer. Phenylthiohydantoin deriva- quantitative release of radioactive peptides (80%),which were tives were separated and quantitated on an Applied Riosystem120A retained on the reversed phase column. Treatment of memanalyzer. braneswith a low concentration of SDS (0.03%) slightly Chemicals"['H]PLP, 4-~0rmyl-~HHjpyridoxal 5"phosphate (3.7 X lo4cpm/nmol), was synthesizedby a published method (20). TLCK- increased the relative content of (H' K')-ATPase in membranes (from36 to 45% as determinedby densitometric scanchymotrypsin,thermolysin,pepsin, V8 protease,andmolecular weight markers were obtained from Sigma.All other chemicals used ning after polyacrylamide gel electrophoresis) and increased were of the highest grade available commercially. the efficiency of chymotrypsin digestion: chymotrypsin diges-
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tion of membranes without SDS treatment released only 50% of the radioactivity. Purification of PLP-binding Peptide-The peptides in the Specific Modification of the Enzyme with PLP-As shown in Fig. 1 (lanes 1 and 2 ) , ['HIPLP was mainly incorporated chymotrypsin digest were separated by HPLC using a linear into (H' K')-ATPase, and the modification was abolished gradient of acetonitrile in the presence of trifluoroacetic acid by the addition of ATP (2 mM) (Fig. 1, lane 3 ) . Consistent (Fig. 3a): the radioactivity was detected only at the position with this finding, ATP at this concentrationcompletely pro- just after the third major peak eluted with about 14% acetotected theenzyme from inhibition by PLP. FITC isknown to nitrile. No more radioactivity was eluted when the column bind to a Lys residue located in the ATP-binding siteof the was washed further with a solvent containing60% solution B enzyme (21), so we examined its effect in prevent.ing modifi- in solution A and then with methanol. These results suggest cation of the enzyme by PLP. After treatment with FITC, the that the radioactivepeak contained most of the modified enzyme was not modified by ["HIPLP (Fig. 1, lane 4 ) . These peptide(s). Radioactivity was not detected in the corresponding position when the chymotryptic peptides from vesicles RESULTS
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1
2
3
4 c
-
116-kDa9768-
45-
Y
origin
-
94-LDa-
94-kDa
' * *
-origin
--
57-LDa 42-
4
-
dye f r o n t
FIG. 1. Protection of (H+ + K+)-ATPase by ATP and FITC from modification with ('HIPLP. Gastric memhrane vesicles (lane 1 ) were modifiedwith 30 p~ ["HIPLP in the ahsence (lane 2) or presence (lane 3 ) of 2 mM ATP. Vesicles pretreated with FITC were alsoincuhatedwith["HIPLP(lane 4 ) . Aliquots of sampleswere examined hy 10% (w/v) SDS-polyacrylamide gel electrophoresis and staining with Coomassie Brilliant Blue (lane 1, 20 pg of protein) or autoradiographv (lanes 2-4, 400 p g of protein). Arrows indicate the positions of molecular mass markers (116 kDa, @-galactosidase;97.4 kDa, phosphorylase h; 68 kDa, bovine serum albumin; 45 kDa, ovalhumin). Details of procedures are described under "Materials and Methods." Incorporation of ["HIPLP into the FITC-bound enzyme was less than 15%of that into untreated enzymeas determined from the radioactivity of gel slices (solubilized with 30% H20?)(3).
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17-dye
front
FIG.2. Chymotryptic digestion of (H+ + K+)-ATPase modified with ["HIPLP. Memhranevesicleswere modified with ["HI PLP and digested with chymotr-ypsin on ice for 15 min as described under"MaterialsandMethods." A portion(400 pg of memhrane protein)wasanalyzed hy SDS-polyacrylamide gel electrophoresis (125) and autoradiography. The weight ratios of chymotrypsin to memhrane protein for lanes1-5 were 0 , 1/200,1/100,1/50, and 1/20, respectively. The molecular mass markers used were bovine serum alhumin (68 kDa), ovalhumin (45 kDa), carbonic anhydrase (29 kDa), soyheantrypsininhihitor(20kDa), and lysozyme (14kDa).The positions of the enzyme hefore chymotrypsin treatment (94 kDa) and the 57-, 42-, and 17-kDa fragments are shownby arrou~s.
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LYS-497of Gastric (Hi K+)-ATPase
8582
0.8
I ”
a
I
hl
“ I Y
20
0
60
40
Retention Time ( m i n ) G 0
20
40
60
80
100
12
Retention Time ( m i n )
FIG. 3. Separation of the labeled peptides by reversedphase HPLC. Membrane vesicles modified with [3H]PLP were incubated with chymotrypsin, and the peptides released were lyophilized and dissolved in water. A portion of the solution (46,000 cpm/ 400 pl) was subjected to HPLC ona CI8 reversed-phase column (Shim-pack CLC-ODS) equilibrated with solution A. The column was washed with solution A for 20 min and then peptides were eluted with a linear gradient of 0-40% (v/v) solution B in solution A. This gradient elution was carried out within 100 min a t a flow rate of 1.0 ml/min. Fractions of 3 ml of eluent were collected, and their radioactivities were measured (histogram). The total radioactivity eluted from the column was35,000 cpm with 21,000cpm in the peak fractions between 56 and 65 min (a). Thesame chromatography was repeated nine times, and the peak fractions between 59 and 62 min were pooled for further purification. An equivalent amount of peptides from vesiclesincubated with [3H]PLPin the presence of ATP (2 mM) was also subjected to HPLC under the same conditions ( b ) . Details of the HPLC were as described under “Materials andMethods.”
incubated with PLP and ATP were applied to the same column (Fig. 3b). Furthermore, a small peak of absorbance at 214 nm (Fig. 3a, indicated by an arrow) was not detectable in the chromatogram of chymotryptic peptides from vesicles incubated with ATP and PLP (Fig. 3b). These results strongly suggested that the radioactive peak in Fig. 3a contained a unique peptide(s) carrying a Lys residue that was modified with PLP. The radioactive fractions were pooled and fractionated on the same column using the second solvent system. Two radioactive peaks (peak I and 11) were eluted with 15 and 16% acetonitrile, respectively (Fig. 4a). No peak corresponding to peak I or I1 was detectable in the chymotryptic digest of vesicles treated with PLP in the presence of ATP (Fig. 4b). Essentially the same results were obtained with three independent membrane preparations. Sequence Determination of Peptides Modified with PLPBoth radioactive peptides were purified further by passage through a reversed phase column and then subjected to gas-
FIG. 4. Rechromatography of radioactive peptides. The radioactive fractions from Fig. 3 were concentrated and dissolved in 10 mM ammonium acetate (pH 6.0) and a portion (28,000 cpm/100 pl) was subjected to HPLCusing the same column as for Fig. 3. Elution with a gradient of0-40% solution D in solution C in 100 min was carried out at a flow rate of1.0 ml/min. The radioactivity in each fraction (3 ml) were monitored (histogram). The total radioactivities recovered in peaks I and II were 8,400 and 12,200 cpm, respectively (a). The same chromatography was repeated five times, and pooled fractions were rechromatographed on Cosmosil5C-18. An equivalent amount of pooled fraction from vesicles incubated with [3H]PLP and ATP was also subjected to HPLC( b ) .
TABLEI Determination of amino acid sequencesof PLP-binding peptides PLP-labeled peptides purified as shown in Figs. 3 and 4 were used for amino acid sequence analysis. The recovery of the amino acid residue (phenylthiohydantoin derivative) identified in each cycle is shown in parentheses. Sequence analyses of peptides from different batches gave the same results. The repetitive yield for peak I1 (Asn(cyc1el)/Asn(cycle 4)) was 95%. Cycle No. Peak I Peak I1 nmol
1
2 3 4 5 6
Ser (0.8) Thr (0.4) Asn (0.3) Pyridoxyl-Lys” Phe (0.2)
Asn (0.7) Ser (1.4) Thr (1.1) Asn (0.6) Pyridoxyl-Lys” Phe (0.4)
Identified as pyridoxyl-Lys, although not determined quantitatively.
phase sequencing (Table I). The sequence of peptide I (peak Z,Fig. 4a) was NH2-Ser-Thr-Asn-Lys-Phe-COOH and thatof peptide I1 (peak ZZ,Fig. 4a) was NH2-Asn-Ser-Thr-Asn-LysPhe-COOH. Peptide I maybe derived from peptide I1 by cleavage of its amino-terminal Asn residue, because chymotrypsin can cleave the carboxyl-terminal side of an Asn residue of a protein (22). The Lys residues in both peptides were modified with PLP, aspyridoxyl-Lys was detected in cycles 4 and 5 for peptides I and 11, respectively. Furthermore, the radioactivity due to [3H]PLP was eluted only in the corre-
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Lys-497 of Gastric (H+ K+)-ATPase
8583
believed to be the residue at the ATP-binding site(21). The close positionalrelationship of these two Lys residues is consistent with the finding that pretreatment of the enzyme with FITC inhibited further modification with PLP, as described above. (iii) ATP hasa protective effect against modification of the enzyme by PLP, but poor substrates such as GTP, CTP, and UTP have no significant protective effect DISCUSSION (3). Furthermore, although ATP and ADP have protective Thepresentresultsdemonstratedthat P L P modified a effects against modification by PLP, phosphate and AMP do specific Lys residue in pig gastric (H' + K+)-ATPase. Two not (3), suggesting that Lys-497 is located near the position peptides modified with [3H]PLP could be solubilized quanti- of the (3-phosphate of adenine nucleotide. This Lys residue tatively from gastric vesicles by chymotrypsin treatment, and locatedbetween phosphorylated Asp (Asp-386) and FITCsequencing studies on the purified peptides indicated that binding Lys residues is conserved in (H' + K')-ATPase of they were derived from residues 494-498 and 493-498, respec- pig (17) and rat (24), the01 subunits of (Na' + K')-ATPases tively, of the enzyme (17) and that Lys-497 was modified with fromvarioussources (25-30) and Ca2'-ATPases of rabbit PLP. The following findings suggested that Lys-497 may be muscle sarcoplasmic reticulum (31,32) and rat brain (33)and located in the catalytic site or in vicinity: its (i) P L P is known human teratoma (34) plasma membranes (Fig. 5). Thus, Lysto have affinity for Lys residues related to the binding sites 497 may have important roles in cation-transporting ATPases of phosphorylatedcompounds (23). (ii) Lys-497 is located of higher organisms. near the Lys-518residue that reacts with FITC, which is Chemicalmodifications with ATP analogues havebeen used to probe the ATP-binding site in E1E2-type ion-trans(H++K+)ATPase Asn-Ser-Thr-Asn-Lys-Phe 5'-(p-Fluorosulfonyl)benzoyl adenosine porting ATPases. (Na++K+)ATPase a Asn-Ser-Thr-Asn-Lys-Tyr modifies a specific Lys residue in theCY subunit of dog kidney Ca*+ATPase a)Asn-Ser-Val-Arg-Lys-Ser (Na' K+)-ATPase (35). Thisresidue is conserved in (H' + K+)- (17, 24), Ca2+-(31-34), and H'-ATPases (36) and corb)Ser-Arg-Asp-Arg-Lys-Ser responds t o Lys-736 of pig gastric (H' + K+)-ATPase (17). FIG. 5. Comparison of the PLP-reactive sequence of pig gastric (H' + K+)-ATPase withthe corresponding regions of Adenosine triphosphopyridoxal also binds to a specific Lys related ion-transporting ATPases. The sequence around the residue of rabbit sarcoplasmic reticulum Ca2+-ATPase (37). PLP-reactive Lys residue of pig gastric (H+ + K+)-ATPase(residues The latter residue, corresponding t o Lys-708 of pig gastric 493-498) (17) was compared with the same region of other ion- (H' + K')-ATPase and Lys-518 that binds FITC, are contransporting ATPases: that of the a subunits of (Na++ K+)-ATPase served in all E1E2-type ion-transporting ATPases so far sefrom pig kidney (25), sheep kidney (26), rat (27), HeLa cells (28), chicken (29), and Torpedo californicaelectric organ (30), and those of quenced (33,34,36). In thisregard, we studied the kineticsof rabbit sarcoplasmic reticulum Ca*+-ATPase( a ) (31, 32) and ratbrain the inhibition of pig gastric (H' + K+)-ATPase with adenosine triphosphopyridoxal, and results were indistinguishable plasma membrane type Ca2+-ATPase( b ) (33, 34) are shown. from those with PLP, suggesting that adenosine triphosphopyridoxal may recognize Lys-497 of (H' + K')-ATPase. It is a$5 K')interesting to know whether the Lys residue of (Na' n ATPase CY subunit corresponding to Lys-497 of the gastric : o 0 enzyme is modified by PLP. Kyte et at. (40) showed that Lys "* - 5 P in a FITC-binding peptide of (Na' + K')-ATPase was modifiedwith PLP, although entirely different conditions were 1 1000 500 used and other Lys residues (unidentified) were also modified. Residue no. The enzyme maytraverse the membrane several times judging from its hydropathy profile (Fig. 6a). Asp-386, Lys-497, Lys518, Lys-708, and Lys-736 are all located in the same large hydrophilic segment, possibly facing the cytoplasm. These residues may form a catalytic site of pig gastric (H' + K')ATPase as shown in theworking model (Fig. 6b) and should be good targets for further studies using site-directed mutagenesis. However, it must be noted that theresidues containing guanidino, sulfhydryl, and carboxyl groups may also be important for the (H' + K')-ATPase activity, as suggested previously (2, 9-16).
sponding cycle. The sequences of the peptides I and I1 were exactly corresponding to residues 494-498 and 493-498, respectively,deducedfrom the sequence of the cDNA clone K')-ATPase(17). Thus, it is codingforpig gastric (H' concluded that PLP specifically modified Lys-497 of the enzyme.
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+
+
.r(
FIG. 6. Hypothetical organization of amino acid residues in the catalytic siteof (H+ K+)-ATPase.a, hydropathy profile (38) of pig gastric (H' + K')-ATPase based on the primary structure determined from the cDNA sequence (17). The averaged hydropathic index of a nonadecapeptide is plotted. Asp-386 (phosphorylation site), Lys-497 (PLP-binding site), Lys-518 (FITC-binding site), Lys-708 (corresponding to theadenosine triphosphopyridoxal (APrPL)-binding site in Ca2+-ATPase(37)), andLys-736 (corresponding to the 5'@-fluorosulfony1)benzoyl adenosine (FSBA)-bindingsite in the a subunit of (Na' + K+)-ATPase(35)) areshown by arrows. b, possible organization of amino acid residues in the catalytic site of pig gastric (H' K+)-ATPase. e, P-sheet; m,a-helix (39).
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Acknowledgments-We, especially M. Tagaya, are grateful to Prof. Toshio Fukui in thisInstitute for encouragement during this work. REFERENCES 1. Faller, L., Jackson, R., Malinowska, D., Mukidjam, E., Rabon,
E., Saccomani, G., Sachs, G., and Smolka, A. (1982) Ann. N . Y. Acad. Sci. 402, 146-163 2. Sachs, G., Chang, H. H., Rabon, E., Schackman, R., Lewin, M., and Saccomani, G. (1976) J . Biol. Chern. 251,7690-7698 3. Maeda, M., Tagaya, M., and Futai, M. (1988)J. Biol. Chern. 263, 3652-3656 4. Wallmark, B., Stewart, H. B., Rabon, E., Saccomani, G., and Sachs, G. (1980) J. Biol. Chern. 2 5 5 , 5313-5319
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Lys-497 of Gastric (H+ K+)-ATPase
5. Pedersen, P. L., and Carafoli, E. (1987) Trends Biochem. Sci. 12, 146-150,186-189 6. Lane, I. K., Kirley, T. L., and Ball, W. J., Jr. (1986) Biochem. Biophys. Res. Commun. 138, 185-192 K. 7. Farley, R. A., and Faller, L. D. (1985) J.Biol. Chem. 260, 3899390 1 8. Walderhaug, M. O.,Post, R. L., Saccomani, G., Leonard, R. T., and Briskin, D. P. (1985) J. Biol. Chem. 260, 3852-3859 691 -695 9. Lee, H. C., Breitbart, H., Berman, M., and Forte, J . G. (1979) Biochim. Biophys. Acta 553, 8125-8132 107-131 10. Lee, H. C., and Forte, J. G. (1980) Biochim. Biophys. Acta 598, 595-605 11. Ray, T. K., and Forte, J . G. (1976) Biochim. Biophys. Acta 443, 451-467 12. Schrijen, J. J., Van G-Luyben, W. A. H. M., De Pont, J.J. H. H. M., and Bonting, S. L. (1981) Biochim. Biophys. Acta 640, 473-486 13. Forte, J. G., Poulter, J. L., Dykstra, R., Rivas, J., and Lee, H. C. (1981) Biochim. Biophys. Acta 644, 257-265 31. 14. Schrijen, J. J., Luyhen, W. A. H. M., De Pont, J. J. H. H. M., and Bonting, S. L. (1980) Biochim. Biophys. Acta 597, 331344 15. Saccomani, G., Barcellona, M. L., and Sachs, G. (1981) J. Biol. Chem. 256, 12405-12410 16. Lorentzon, P., Jackson, R., Wallmark, B., and Sachs, G. (1987) Biochim. Biophy~.Acta 897, 41-51 17. Maeda, M., Ishizaki, J., and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157,203-209 18. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 19. Laemmli, U. K. (1970) Nature 227,680-685 20. Raibaud, O., and Goldberg, M. E. (1974) FEBS Lett. 40, 41-44 21. Jackson, R. J.,Mendlein,J., andSachs, G. (1983) Biochim. Biophys. Acta 731,9-15 22. Markland, F. S..Kreil, G., Ribadeau-Dumas, B., and Smith, E. L. (1966) J. Bid. Chem. 241,4642-4664 148 23. Means, G. E., and Feeney, R. E. (1971) J.Biol. Chem. 246,55325533
24. Shull, G. E., and Lingrel, J. B. (1986) J. Biol. Chem. 261,1678816791 25. Ovchinnikov, Y. A,, Modyanov, N. N., Broude, N. E., Petrukhin, E., Grishin, A. V., Arzmazova, N. M., Aldanova, N. A., Monastyrskaya, G. S., and Sverdlov, E. D. (1986) FEBS Lett. 201,237-245 26. Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Nature 316, 27. Shull, G. E., Greeb, J., and Lingrel, J. B. (1986) Biochernistry25, 28. Kawakami, K., Ohta, T., Nojima, H., and Nagano, K. (1986) J. Biochem. (Tokyo) 100, 389-397 29. Takeyasu, K., Tamkun, M. M., Renaud, K. J., and Fambrough, D. M. (1988) J. Biol. Chem. 263, 4347-4354 30. Kawakami, K., Noguchi, S., Noda, M., Takahashi, H., Ohta, T., Kawamura, M., Nojima, H., Nagano, K., Hirose, T., Inayama, S., Hayashida, H., Miyata, T., and Numa, S. (1985) Nature 316,733-736 MacLennan, D. H., Brandl, C. J., Korczak, B., and Green, N. M. (1985) Nature 316, 696-700 32. Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H. (1986) Cell 44,597-607 33. Shull, G. E., and Greeb, J. (1988) J. Biol. Chem. 263,8646-8657 34. Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. F., Fischer, R., Heim, R., Vogel, G., Mathews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988) J . Biol. C&m. 263, 1415214159 35. Ohta, T., Nagano, K., and Yoshida, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2071-2075 36. Serrano, R. (1988) Biochim. Biophys. Acta 947, 1-28 37. Yamamoto, H., Tagaya, M., Fukui, T., and Kawakita, M. (1988) J . Biochem. (Tokyo) 103,452-457 38. Kyte, and J., Doolittle, R. F.(1982) J. Mol. Biol. 157, 105-132 39. Chou, P. Y., and Fasman, G. D. (1978) Adu. Biochem. 47. 4540. Kyte, J., Xu, K.-Y., and Bayer, R. (1987) Biochemistry 26,83508360
