Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents
1997; Elsevier BV; Volume: 272; Issue: 36 Linguagem: Inglês
10.1074/jbc.272.36.22438
ISSN1083-351X
AutoresMarie Besancon, Alex Simon, George Sachs, Jai Moo Shin,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoThe vesicular gastric H,K-ATPase catalyzes an electroneutral H for K exchange allowing acidification of the intravesicular space. There is a total of 28 cysteines present in the α subunit of the gastric H,K-ATPase, of which 10 are found in the predicted transmembrane segments and their connecting loop, and 9 are present in the β subunit, of which 6 are disulfide-linked. To determine which of these was accessible to extracytoplasmic attack, the enzyme was inhibited by four different substituted 2-pyridylmethylsulfinyl benzimidazoles, 5-methoxy-2-[(4-methoxy-3,5-dimethyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole (omeprazole), 2-[(4-trifluoroethoxy-3-methyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole (lansoprazole), 5-difluoromethoxy-2-[3,4-methoxy-2-pyridyl)methylsulfinyl]-1H-benzimidazole (pantoprazole), and 2-[(4-(3-methoxypropoxy)-3-methyl)-2-pyridyl)methylsulfinyl]-1H-benzimidazole (rabeprazole), under acid transporting conditions. All of these compounds are weak bases that accumulate in the acidic space generated by the pump and undergo an acid catalyzed rearrangement to a cationic sulfenamide, which forms disulfides with accessible cysteines. The relative rates of acid activation of these compounds corresponded to the relative rates of inhibition of ATPase activity and acid transport. Fragmentation of the enzyme by trypsin followed by SDS-polyacrylamide gel electrophoresis showed that omeprazole bound covalently to one of the two cysteines in the domains containing the fifth and sixth transmembrane segments and their extracytoplasmic loop and to cysteine 892 in the loop between the seventh and eighth transmembrane segments, but inhibition correlated with the reaction with cysteines in the fifth and sixth domain. Lansoprazole bound to the cysteines in these two domains as well as to cysteine 321 toward the extracytoplasmic end of the third transmembrane segments. Pantoprazole bound only to either cysteine 813 or 822 in the fifth and sixth transmembrane region. The inhibition of Rabeprazole correlated also with its binding to this part of the protein, but this compound continued to bind after full inhibition, eventually binding also to cysteines 321 and 892. No binding was found to any of the cysteines in the seventh to tenth transmembrane segments. Thermolysin digestion of the isolated omeprazole-labeled fifth and sixth transmembrane pair showed that cysteine 813 was the site of labeling. It is concluded that binding of these sided reagents to cysteine 813 in the loop between transmembrane (TM)5 and TM6 is sufficient for inhibition of ATPase activity and acid transport by the gastric acid pump. Of the 10 cysteines present in the membrane and extracytoplasmic domain, only three are exposed sufficiently to allow reactivity with these cationic thiol reagents. The binding to cysteine 813 defines the location of the extracytoplasmic loop between TM5 and TM6 and places the carboxylic acids 820 and 824 conserved between the gastric H,K- and the Na,K-ATPases in TM6, consistent with their assumed role in cation binding. The vesicular gastric H,K-ATPase catalyzes an electroneutral H for K exchange allowing acidification of the intravesicular space. There is a total of 28 cysteines present in the α subunit of the gastric H,K-ATPase, of which 10 are found in the predicted transmembrane segments and their connecting loop, and 9 are present in the β subunit, of which 6 are disulfide-linked. To determine which of these was accessible to extracytoplasmic attack, the enzyme was inhibited by four different substituted 2-pyridylmethylsulfinyl benzimidazoles, 5-methoxy-2-[(4-methoxy-3,5-dimethyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole (omeprazole), 2-[(4-trifluoroethoxy-3-methyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole (lansoprazole), 5-difluoromethoxy-2-[3,4-methoxy-2-pyridyl)methylsulfinyl]-1H-benzimidazole (pantoprazole), and 2-[(4-(3-methoxypropoxy)-3-methyl)-2-pyridyl)methylsulfinyl]-1H-benzimidazole (rabeprazole), under acid transporting conditions. All of these compounds are weak bases that accumulate in the acidic space generated by the pump and undergo an acid catalyzed rearrangement to a cationic sulfenamide, which forms disulfides with accessible cysteines. The relative rates of acid activation of these compounds corresponded to the relative rates of inhibition of ATPase activity and acid transport. Fragmentation of the enzyme by trypsin followed by SDS-polyacrylamide gel electrophoresis showed that omeprazole bound covalently to one of the two cysteines in the domains containing the fifth and sixth transmembrane segments and their extracytoplasmic loop and to cysteine 892 in the loop between the seventh and eighth transmembrane segments, but inhibition correlated with the reaction with cysteines in the fifth and sixth domain. Lansoprazole bound to the cysteines in these two domains as well as to cysteine 321 toward the extracytoplasmic end of the third transmembrane segments. Pantoprazole bound only to either cysteine 813 or 822 in the fifth and sixth transmembrane region. The inhibition of Rabeprazole correlated also with its binding to this part of the protein, but this compound continued to bind after full inhibition, eventually binding also to cysteines 321 and 892. No binding was found to any of the cysteines in the seventh to tenth transmembrane segments. Thermolysin digestion of the isolated omeprazole-labeled fifth and sixth transmembrane pair showed that cysteine 813 was the site of labeling. It is concluded that binding of these sided reagents to cysteine 813 in the loop between transmembrane (TM)5 and TM6 is sufficient for inhibition of ATPase activity and acid transport by the gastric acid pump. Of the 10 cysteines present in the membrane and extracytoplasmic domain, only three are exposed sufficiently to allow reactivity with these cationic thiol reagents. The binding to cysteine 813 defines the location of the extracytoplasmic loop between TM5 and TM6 and places the carboxylic acids 820 and 824 conserved between the gastric H,K- and the Na,K-ATPases in TM6, consistent with their assumed role in cation binding. The gastric H,K-ATPase is a member of the P type ATPase family. Transport of H3O+ outward in exchange for K+ transport inward is coupled to a cycle of phosphorylation and dephosphorylation. In conjunction with parallel K+ and Cl− conductances, this ATPase is responsible for the elaboration of HCl into the secretory canaliculus of the parietal cell or into isolated purified gastric vesicles, the enclosed space reaching a pH of about 1 (1Rabon E. Reuben M.A. Annu. Rev. Physiol. 1990; 52: 321-344Crossref PubMed Scopus (154) Google Scholar). A number of chemical reagents have been useful in analyzing several aspects of structure function in this P type ATPase and the Na,K-TPase (1Rabon E. Reuben M.A. Annu. Rev. Physiol. 1990; 52: 321-344Crossref PubMed Scopus (154) Google Scholar, 2Kaplan J.H. Soc. Gen. Physiol. Ser. 1991; 46: 117-128PubMed Google Scholar). For example, DCCD 1The abbreviations used are: DCCD, dicyclohexylcarbodiimide; PVDF, polyvinylidene difluoride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NEM, N-ethylmaleimide; omeprazole, 5-methoxy-2-[(4-methoxy-3,5-dimethyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole; lansoprazole, 2-[(4-trifluoroethoxy3-methyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole; pantoprazole, 5-difluoromethoxy-2-[3,4-methoxy-2-pyridyl)methylsulfinyl]-1H-benzimidazole; rabeprazole, 2-[(4-(3-methoxypropoxy)-3-methyl)-2-pyridyl)methylsulfinyl]-1H-benzimidazole; TM, transmembrane segment.1The abbreviations used are: DCCD, dicyclohexylcarbodiimide; PVDF, polyvinylidene difluoride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NEM, N-ethylmaleimide; omeprazole, 5-methoxy-2-[(4-methoxy-3,5-dimethyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole; lansoprazole, 2-[(4-trifluoroethoxy3-methyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole; pantoprazole, 5-difluoromethoxy-2-[3,4-methoxy-2-pyridyl)methylsulfinyl]-1H-benzimidazole; rabeprazole, 2-[(4-(3-methoxypropoxy)-3-methyl)-2-pyridyl)methylsulfinyl]-1H-benzimidazole; TM, transmembrane segment. is a hydrophobic reagent that reacts with a carboxylic group in the membrane domain of either pump in a K protectable manner (3Goldshleger R. Tal D.M. Moorman J. Stein W.D. Karlish S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6911-6915Crossref PubMed Scopus (59) Google Scholar, 4Rabon E.C. Hoggatt M. Smillie K. J. Biol. Chem. 1996; 271: 32137-32146Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) and thereby interfering not only with ATPase activity but also with Rb occlusion. Fluorescein isothiocyanate reacts with a cytoplasmic lysine in these pumps, providing a fluorescent marker (5Karlish S.J.D. J. Bioenerg. Biomembr. 1980; 12: 111-136Crossref PubMed Scopus (236) Google Scholar, 6Rabon E.C. Bassilian S. Sachs G. Karlish S.J.D. J. Biol. Chem. 1990; 265: 19594-19599Abstract Full Text PDF PubMed Google Scholar) for Na- and K-induced conformations. Thiol reagents have been used to advantage in the Na,K-ATPase to define reactive cysteines in the membrane or extracytoplasmic domain (7Esmann M. Hideg K. Marsh D. Biochim. Biophys. Acta. 1992; 1112: 215-225Crossref PubMed Scopus (9) Google Scholar, 40Lingrel J.B Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). Sided reagents are relatively rare. Ouabain, a partially K-competitive inhibitor of the Na,K-ATPase, binds to the extracytoplasmic surface of the Na,K-ATPase. Since this ligand is non-covalent, mutagenesis has been used to establish that it binds or interacts with the first, second, fifth, and sixth transmembrane domains (8Feng J. Lingrel J.B. Biochemistry. 1994; 33: 4218-4224Crossref PubMed Scopus (63) Google Scholar, 9Palasis M. Kuntzweiler T.A. Arguello J.M. Lingrel J.B J. Biol. Chem. 1996; 271: 14176-14182Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The essential contribution of the H,K-ATPase to acid secretion by the stomach has resulted in the synthesis of compounds that inhibit this enzyme selectively (10Sachs G. Shin J.M. Briving C. Wallmark B. Hersey S. Ann. Rev. Pharmacol. Toxicol. 1995; 35: 277-305Crossref PubMed Scopus (355) Google Scholar). One class consists of substituted 2-(2-pyridylmethylsulfinyl)-1H-benzimidazoles. These compounds are protonatable weak bases of pK a about 4.0 or 5.0. This pK a and the membrane permeability of the unprotonated form result in their selective accumulation in the acidic space of the secretory canaliculus of the active parietal cell or in the acid transporting gastric-derived vesicles studied here. Following accumulation, the compounds undergo an acid-catalyzed rearrangement to a cationic sulfenamide in the extracytoplasmic space (11Lindberg P. Nordberg P. Alminger T. Brandstrom A. Wallmark B. J. Med. Chem. 1986; 29: 1327-1329Crossref PubMed Scopus (325) Google Scholar, 12Senn-BillFinger J. Kruger U. Sturm E. Figala V. Klemm K. Kohl B. Rainer G. Schaefer H. Blake T.J. Darkin D.W. Ife R.J. Leach C.A. Mitchell R.C. Pepper E.S. Slater C.J. Vine N.J. Huttner G. Zsolnai L. J. Org. Chem. 1987; 52: 4582-4592Crossref Scopus (58) Google Scholar). This cationic sulfenamide is relatively membrane impermeant and is able to react with thiol groups within the catalytic subunit of the H,K-ATPase to form relatively stable disulfides (13Besancon M. Shin J.M. Mercier F. Munson K. Miller M. Hersey S. Sachs G. Biochemistry. 1993; 32: 2345-2355Crossref PubMed Scopus (128) Google Scholar, 24Lorentzon P. Jackson R. Wallmark B. Sachs G. Biochim. Biophys. Acta. 1987; 897: 41-51Crossref PubMed Scopus (101) Google Scholar, 30Keeling D.J. Fallowfield C. Underwood A.H. Biochem. Pharmacol. 1987; 36: 339-344Crossref PubMed Scopus (58) Google Scholar). Since the thiophilic cation is formed on the outside surface of the ATPase, it is likely that the cysteines reacting are accessible only from the extracytoplasmic face of the pump. Therefore, these benzimidazoles, under conditions of acid transport by the ATPase, (a) can provide structural information by defining the cysteines that are accessible from the outside surface of the pump and (b) can provide functional information about the region of this surface to which they bind since they inhibit ATPase activity, transport, and all partial reactions of the enzyme (13Besancon M. Shin J.M. Mercier F. Munson K. Miller M. Hersey S. Sachs G. Biochemistry. 1993; 32: 2345-2355Crossref PubMed Scopus (128) Google Scholar). Of the 28 cysteines in the α subunit according to the 10-transmembrane segment model, ten are predicted to be in the membrane or in the extracytoplasmic space and one or more of these are implicated in inhibition of the ATPase by these reagents (14Bamberg K. Sachs G. J. Biol. Chem. 1994; 269: 16909-16919Abstract Full Text PDF PubMed Google Scholar). The membrane domain of this ion pump, as for the sarcoplasmic reticulum Ca2+-ATPase, the Na,K-ATPase, and the Mg2+-ATPase of Salmonella typhimurium as well as the H+-ATPase of Neurospora orSaccharomyces, is thought to consist of ten transmembrane segments connected by five extracytoplasmic loops (15MacLennan D.H. Brandl C.J. Korczak B. Green N.M. Nature. 1985; 316: 696-700Crossref PubMed Scopus (803) Google Scholar, 16Karlish S.J.D. Goldshleger R. Jorgensen P.L. J. Biol. Chem. 1993; 268: 3471-3478Abstract Full Text PDF PubMed Google Scholar, 17Smith D.L. Tao T. Maguire M.E. J. Biol. Chem. 1993; 268: 22469-22479Abstract Full Text PDF PubMed Google Scholar, 18Padmanabha K.P. Petrov V. Ambesi A. Rao R. Slayman C.W. Symp. Soc. Exp. Biol. 1994; 48: 33-42PubMed Google Scholar, 19Mahanty S.K. Scarborough G.A. J. Biol. Chem. 1996; 271: 367-371Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Mutagenesis studies carried out largely on the sarcoplasmic reticular Ca2+-ATPase and the Na,K-ATPase place ion binding sites within this membrane domain, in particular in the fourth, fifth, sixth, and eighth transmembrane segments (9Palasis M. Kuntzweiler T.A. Arguello J.M. Lingrel J.B J. Biol. Chem. 1996; 271: 14176-14182Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 20Kuntzweiler T.A. Arguello J.M. Lingrel J.B J. Biol. Chem. 1996; 271: 29682-29687Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 21Johnson C.L. Kuntzweiler T.A. Lingrel J.B. Johnson C.G. Wallick E.T. Biochem. J. 1995; 309: 187-194Crossref PubMed Scopus (21) Google Scholar). The conserved carboxylic acids in the putative fifth and sixth transmembrane segments are placed within these segments because mutagenesis affects ion transport. Proof that they are in the membrane segments rather than in the extracytoplasmic loop has not been provided. Four substituted 2-(2-pyridyl-methylsulfinyl)-1H-benzimidazoles were used in this study, namely omeprazole, lansoprazole, pantoprazole, and rabeprazole, to identify the luminally accessible cysteines and those relevant to inhibition of the ATPase by these compounds. Their structures are shown in Fig. 1, as is their probable chemical mechanism of activation. They are shown as accumulating in acid due to protonation. This is followed by acid activation to the sulfenamide, which in turn results in binding to cysteines of the gastric H,K-ATPase. The sulfenamide is a permanent cation, restricting its membrane permeability and is, therefore, an extracytoplasmic thiol reagent. The compounds differ in their acid stability. Pantoprazole is the most stable at neutral pH, omeprazole and lansoprazole have about equal stability, and rabeprazole is the least stable at neutral pH. All four compounds convert to the sulfenamide more rapidly at pH 3.0 and below but retain their relative rates of conversion. Thet½ of conversion of omeprazole or lansoprazole at pH 5.0 is about 0.3 h, whereas that of pantoprazole is about 1.2 h and rabeprazole is about 0.01 h (35Singh P. Sharma R.C. Ojha T.N. Drug Design Deliv. 1991; 7: 131-138PubMed Google Scholar). 2J. Senn-Billfinger, personal communication.2J. Senn-Billfinger, personal communication. After acid activation, the compounds are unstable at neutral pH, converting to the inactive sulfide and other substances (22Brandstrom A. Lindberg P. Bergman N-A. Alminger T. Ankner K. Junggren U. Lamm B. Nordberg P. Erickson M. Grundevik I. Hagin I. Hoffmann K-J. Johansson S. Larsson S. Lofberg I. Ohlson K. Persson B. Skinberg I. Tekenbergs-Hjelte L. Acta Chem. Scand. 1989; 43: 536-548Crossref Google Scholar), but are relatively stable in acidic solution. The effect of these four compounds was studied on vesicular preparations of the hog gastric H,K-ATPase under acid transporting conditions, in terms of the rate and level of inhibition of enzyme activity, acid transport, and the sites of labeling of the enzyme. The rate of inhibition of ATPase activity and of proton transport correlated with the degree of acid stability, rabeprazole being faster than omeprazole, which was equal to lansoprazole with pantoprazole being slowest. Labeling of different cysteines was found. In general, the first cysteines labeled were those contained within the fifth and sixth transmembrane domains and the connecting extracytoplasmic loop, and this labeling corresponded to the onset of inhibition. Pantoprazole labeled only these cysteines, omeprazole labeled mainly the same region but, in addition, the loop between the seventh and eighth transmembrane segments. Lansoprazole labeled, in addition to the cysteines labeled by omeprazole, the cysteine present in the domain contained within the third and fourth transmembrane segments and the connecting loop. Rabeprazole labeled the fifth and sixth transmembrane segment, resulting in full inhibition of ATPase activity and acid transport but continued to label the enzyme after full inhibition with a pattern of labeling eventually similar to that of lansoprazole. Thermolysin digestion of the isolated omeprazole-labeled tryptic fragment followed by separation and sequencing of the peptide labeled with omeprazole showed that cysteine 813 was the cysteine reacting in the TM5/TM6 domain. Hog gastric vesicles were prepared as described previously (23Rabon E.C. Im W.B. Sachs G. Methods in Enzymol. 1988; 157: 649-654Crossref PubMed Scopus (40) Google Scholar). The ion tight fraction layering on top of a 7.5% Ficoll solution in 250 mm sucrose on the density gradient was used in all the experiments. The average basal activity in the experiments described here was 5 μmol of ATP hydrolyzed/mg of protein/h. 10 μmol of ATP/mg/h was hydrolyzed in the presence of 20 mm KCl and 120 μmol/mg/h in the presence of KCl and nigericin or in the presence of 100 mm NH4Cl. Accordingly, 90% or more of the vesicles were ion tight. K+-stimulated activity with 20 mm KCl in the absence of ionophores is due to leaky vesicles. The ATPase activity in the presence of K+ plus nigericin or of 100 mmNH4Cl was considered to reflect the total ATPase activity (24Lorentzon P. Jackson R. Wallmark B. Sachs G. Biochim. Biophys. Acta. 1987; 897: 41-51Crossref PubMed Scopus (101) Google Scholar), whereas the difference between the latter measurements and the K+-stimulated ATPase in the absence of ionophore reflects the contribution of ion tight vesicles. NH4+ activates the ATPase by permeating as NH3 and then forming NH4+, which is then transported by the H,K-ATPase out of the vesicles as a K+ surrogate resulting in cycling of the enzyme without the formation of a proton gradient (25Sachs G. Rev. Physiol. Biochem. Pharmacol. 1977; 79: 133-162Crossref PubMed Google Scholar). Pi released was measured by the method of Yoda and Hokin (26Yoda A. Hokin L.E. Biochem. Biophys. Res. Commun. 1970; 40: 880-886Crossref PubMed Scopus (190) Google Scholar) and protein by the Lowry method (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Acidification of the gastric vesicles was measured by the quenching of acridine orange as described previously (28Rabon E. Chang H. Sachs G. Biochemistry. 1978; 17: 3345-3353Crossref PubMed Scopus (60) Google Scholar). Briefly, the vesicles at 10 μg/ml were suspended in a medium containing 250 mm sucrose, 150 mm KCl, 1 μm acridine orange, 5 mm Tris/HCl buffer, pH 6.8, and 1 μg/ml valinomycin. The inhibitors, when present, were added at 20 μm final concentration. Transport was initiated by the addition of 2 mm MgATP, pH 6.8, and the fluorescence of acridine orange was measured as a function of time in a Spex fluorimeter with excitation at 480 nm and emission at 530 nm. Inhibition of the ATPase by the compounds was evaluated during the labeling reaction and also in the absence of radioactive material but under otherwise identical conditions. In summary, the enzyme under acid transporting conditions was incubated with the drug, and samples were taken at time zero and various other time points for measurement of labeling and of enzyme activity. Inhibition of enzyme activity was obtained by comparing the phosphate present in the samples at any given time point with the phosphate released following the addition of ATP and NH4+ to the samples as described previously (13Besancon M. Shin J.M. Mercier F. Munson K. Miller M. Hersey S. Sachs G. Biochemistry. 1993; 32: 2345-2355Crossref PubMed Scopus (128) Google Scholar, 24Lorentzon P. Jackson R. Wallmark B. Sachs G. Biochim. Biophys. Acta. 1987; 897: 41-51Crossref PubMed Scopus (101) Google Scholar). Thus, vesicles were added to a solution containing 250 mmsucrose, 150 mm KCl, 5 mm Tris/HCl, pH 6.8, and 1 μg/ml valinomycin at 37 °C to a final protein concentration of 100–200 μg/ml. The solutions contained 10 μm14C-labeled compound (lansoprazole, pantoprazole, and rabeprazole) or 3H-labeled compound (omeprazole). The specific activity of the [3H]omeprazole in the experiment illustrated was 315 cpm/pmol, that of [14C]lansoprazole was 43 cpm/pmol, and that of [14C]pantoprazole was 50 cpm/pmol, and [14C]rabeprazole had a specific activity of 82 cpm/pmol in the experiment illustrated in the figure. MgATP at pH 6.8 was added to the reaction mixture at 2.5 mm to start the inhibition and two 10-μl samples were taken to measure enzyme activity at zero time. After the addition of ATP, four 10-μl aliquots were taken at 5, 10, 15, 30, and 45 min to measure residual enzyme activity. The inhibitory reaction was stopped by 5-fold dilution of the sample in ice-cold buffer containing 20 mm Tris/HCl, pH 7.0, 1 mmEDTA, and 250 mm sucrose and 5 mm NEM. Residual ATPase activity was measured in the absence and presence of 100 mm NH4Cl as described above with a correction for the Pi released during the prior incubation period. One set of duplicates was used for measurement of Pi that had been released during the inhibition reaction, and another set of two 10-μl samples was used for measurement of residual ATPase activity. These samples were added to assay medium on ice. An additional 2 mm MgATP was added to start the reaction at 37 °C. ATPase activity was measured in a solution of 40 mmTris/HCl, pH 7, 100 mm NH4Cl, and 250 mm sucrose for 15 min. The ATPase activity remaining at each successive time point was obtained from the additional Pi released in the second incubation. At either 5 or 10 min and at 30 or 45 min, half or the rest of the reaction mixture was removed to define the labeling of the enzyme. This sample was diluted 5-fold with ice-cold 20 mm Tris/HCl, pH 7.4, 250 mm sucrose, 5 mm NEM, and 1 mm EDTA and spun in a Beckman L5 centrifuge using a Ti-35 rotor at 100,000 × g for 60 min. The pellet was washed twice with 20 mm Tris/HCl, 250 mm sucrose, pH 7.0, by centrifugation and resuspended in the same buffer to give a final protein concentration of 1–3 mg/ml. The sample was frozen and stored at −80 °C until further use. 150 μg of labeled vesicles were digested at a protein to trypsin ratio of 5:1 at 37 °C for 5 min. The reaction was stopped by the addition of 5-fold excess trypsin inhibitor, and the samples were placed immediately on ice. The digested membranes were pelleted in a Beckman Airfuge at 28 psi. 10 μl of buffer containing 20 mm Tris/HCl, pH 7.4, and 5 mm NEM as above was added to the pellet along with 10 μl of 4% CHAPS detergent to dissolve the pellet. 80 μl of ice-cold methanol was added to precipitate the protein, and the mixture was kept on ice for 10 min prior to recentrifugation in the Beckman airfuge. The pellet was now dissolved in sample buffer containing 25% volume of 0.3m Tris buffer, pH 6.8, 10% SDS, 40% sucrose, and 0.25% bromphenol blue and 75% volume of 20 mm Tris/HCl with 5 mm NEM. The peptides were separated by SDS-PAGE. The membrane digests dissolved in 25% volume of sample buffer were placed on top of a 10% (34:1 acrylamide/methylene bisacrylamide) to 21% (17:1 acrylamide/methylene bisacrylamide) 1.5-mm gradient slab gel using the Tricine buffer method (29Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10457) Google Scholar). The gel was run in the cold room (4 °C) for 18 h at 90 V constant voltage along with a lane for prestained molecular mass (Bio-Rad, 106–18 kDa) standards and CNBr fragments of horse myoglobin (Sigma, 17–2.5 kDa). In every case, a duplicate lane was run to provide material for sequencing as well as for either counting or autoradiography. Reducing agents were absent in all experiments since these remove the bound benzimidazole. Standard curves of ln(M r) as a function of relative mobility were used to estimate the M rof the peptide products of digestion. The accuracy of theM r weight determination appeared to be within 10% based on predicted tryptic cleavage sites within the primary sequence of catalytic subunit of the enzyme. The peptides were transferred electrophoretically to PVDF membranes (Millipore) for 18–24 h in the cold room (4 °C) in a tank transfer apparatus at 120 mA constant current, in a transfer buffer of 150 mm glycine, 20 mm Tris, and 20% methanol. A sandwich of three sheets of Whatman 3-mm filter paper was placed on either side of the gel, which had a prewetted PVDF membrane on the anode side. After transfer, the blots were rinsed twice in distilled water and stained with 0.1% Coomassie Blue in 10% glacial acetic acid and 45% methanol. The PVDF membrane was sprayed with EN3HANCE (DuPont) and exposed to x-ray film in the cold for 8–14 days. The film was developed, and the bands corresponding to the radioactivity on the autoradiogram were cut out, a portion was taken for counting, and the rest were subjected to microsequencing in a microsequencing facility. [3H]Omeprazole-labeled vesicles were extensively digested. 0.8 mg of [3H]omeprazole-labeled vesicles were incubated with 0.2 mg of trypsin in a buffer (1 ml) composed of 0.25 msucrose, 50 mm Tris/HCl, pH 8.2, for 30 min. The reaction was stopped by adding 2 mg of soybean trypsin inhibitor. The membrane digest was spun in a Beckman L5 centrifuge using a Ti-65 rotor at 100,000 × g for 60 min. The pellet was dissolved in a buffer (0.8 ml) composed of 20 mm Tris/HCl, pH 6.7, 0.2 mm fluorescein-5-maleimide, and 0.5% SDS, and electrophoresed as described above. The lowest fluorescent band at 5.5–6.2 kDa range, containing the [3H]omeprazole-labeled TM5/TM6 segment, was sliced from the gel. The labeled peptide was electroeluted using Bio-Rad Electroeluter Model 422 equipped with a Membrane Caps (molecular cut-off 3.5 kDa). Electroelution was carried out in a buffer composed of 30 mm Tris/HCl, pH 8.0, 0.03% SDS at 120 V constant voltage for 3 h. It was necessary to reduce the concentration of SDS and salt of the eluate for further digestion. The electroelution buffer was carefully removed and replaced with a new buffer composed of 10 mm Tris/HCl, pH 8.0, 0.01% SDS. Electrodialysis was carried out at 100 V constant voltage for 1 h. The eluate was separated and diluted by adding 2-fold excess volume of 10 mm Tris/HCl, 6 mm CaCl2, pH 8.0. The eluate was divided into two portions. One portion served as a control, and the other portion was digested with 10 μg of thermolysin (Sigma, Protease Type X) at room temperature for 20 h. After digestion, samples were concentrated up to 90 μl by Speed-Vac and combined with 10 μl of 2 m sucrose and 0.25% bromphenol. Samples were electrophoresed using a 16.5% (17:1 acrylamide/methylene bisacrylamide) 1.5-mm gradient slab Tricine gel (29Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10457) Google Scholar). Each lane had 50 μl of samples. The gel was run in the cold room (4 °C) for 18 h at 90 V constant voltage, along with a lane for CNBr fragments of horse myoglobin (Sigma, 17–2.5 kDa). The peptides were transferred electrophoretically to PVDF membranes (Bio-Rad, 0.2 μm pore size) for 6 h in the cold room (4 °C) in a tank transfer apparatus (Idea Scientific Company) at 12 V constant voltage, in a transfer buffer of 15 mm CHAPS/NaOH, pH 11.0, and 10% ethanol. The PVDF membrane was rinsed with distilled water. One lane was sliced for radioactivity counting and the other lane was for microsequencing. With all the compounds tested, 10 mm dithiothreitol removed all counts found in the SDS-solubilized membranes showing that the stable labeling observed was due to the presence of a disulfide linkage between the compound and the enzyme. Omeprazole and radioactive omeprazole were a gift of Astra Hassle, Sweden; lansoprazole and radioactive lansoprazole were a gift from Takeda, Japan; pantoprazole and labeled pantoprazole were a gift of Byk Gulden, Germany; and rabeprazole and labeled rabeprazole (E3810) were a gift of Eisai Ltd., Tokyo, Japan. All other chemicals were analytical grade or better. In the absence of ATP, there was some inhibition of enzyme activity as expected from the p
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