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The orientation of the carboxy-terminal pair of tyrosines of the Na+/K(+)-ATPase alpha-subunit with respect to the plane of the plasma membrane was determined. The approach was based on lactoperoxidase-catalysed radioiodination of the tyrosine residues accessible on the surface of the enzyme molecule in intact cells of a pig kidney embryonic cell line and those accessible in a broken plasma membrane fraction and in isolated membrane-bound Na+/K(+)-ATPase. The labeled alpha-subunit was isolated by SDS gel electrophoresis followed by electroblotting. Then the COOH-terminal amino acids were hydrolyzed by carboxypeptidases B and Y. Radioactivity and quantitative analysis of the protein and released amino acids showed that the COOH-terminal tyrosine residues of the alpha-subunit were only accessible to modification only when lactoperoxidase had access to the inner side of the plasma membrane. Therefore, the COOH-terminus of the Na+/K(+)-ATPase alpha-subunit is located on the cytoplasmic surface of the pump molecule and its polypeptide chain must have an even number of transmembrane segments.

Cholecystokinin stimulates pancreatic zymogen secretion by binding with high affinity to a receptor on the pancreatic acinar cell. This receptor has been cloned and shown to be a CCK-A subtype. CCK also stimulates pepsinogen secretion from the gastric chief cell with high affinity. Using polymerase chain reaction with primers from the known sequence of the rat pancreatic CCK-A receptor cDNA, we prepared a 600 bp product from rat and rabbit stomach cDNA. From Southern analysis these represented a fragment of a gastric CCK-A receptor. PCR was then used to amplify a rabbit λZAP II gastric epithelial cDNA library with the same primers, and the product was identified by sequencing as representing a CCK-A receptor fragment. When this PCR product was used to screen the library, ten positive clones were identified in a screening of 4 · 105 plaques, and several of these were sequenced. All had essentially the same sequence contained within 2 of these clones consisted of 427 amino acids and was 92% homologous (87% identity) to the known rat pancreatic CCK-A sequence but only 43% homologous to the gastric CCK-B sequence. The cDNA was subcloned into a pcDNA1 expression vector and transiently expressed in the human embryonic kidney cell line, HK 293. The responses of intracellular Ca2+ in these transfected cells to CCK and gastrin were monitored using video imaging. On the average 40% of the cells responded to CCK-8 by a transient elevation of [Ca2+]i followed by a steady state plateau. CCK was a high and gastrin a low affinity ligand for this signal, corresponding to the actions of these ligands on pepsinogen secretion from chief cells and somatostatin release from D cells. Hence from sequence and second messenger responses, the clone represents the CCK-A receptor presumably responsible for pepsinogen secretion by gastric chief cells and somatostatin release from gastric D cells.

Several antibodies against the gastric H+/K(+)-ATPase were analysed for the topological and sequence location of their epitopes. Topological mapping was done by comparing indirect immunofluorescent staining in intact and permeabilised rat parietal cells. Epitope definition was by Western analysis of intact and of trypsin or V8-proteinase-fragmented hog gastric ATPase combined with N-terminal sequencing of the fragments; by Western analysis of fragments of rabbit alpha subunit expressed in Escherichia coli; by analysis of rabbit alpha and beta subunits expressed in baculovirus-transfected SF 9 cells and by ELISA assay of synthetic octamers of one region of the hog alpha subunit. It was confirmed that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic region between M4 and M5, close to the ATP-binding domain. The major epitope for monoclonal antibody mAb 12-18 was also in this region, but a second epitope was confirmed to be present in the M7/M8 region. The monoclonal antibody, mAb 146-14, was shown to recognise an extracytoplasmic epitope dependent on intact disulfide bonds, present in the rat and the rabbit, but absent in the hog beta subunit, due to non-conservative amino-acid substitutions. This antibody also recognised an epitope present in the alpha subunit of the H+/K(+)-ATPase at the M7 extracytoplasmic interface, perhaps indicating structural association of these two regions. The polyclonal antibody, pAb39, raised against the C-terminal portion of the enzyme, reacted only with the cytoplasmic surface of the H+/K(+)-ATPase, showing that the alpha subunit of the enzyme has an even number of membrane spanning segments.

We have cloned and sequenced a cDNA for the rabbit gastric proton-potassium pump (H+/K(+)-ATPase) alpha-subunit. The deduced peptide contains 1035 amino acids (Mr 114,201) and shows 97% sequence identity with the respective rat and hog proteins. A monoclonal antibody 146-14 has been shown previously to react with the extracytoplasmic side of the catalytic H+/K(+)-ATPase subunit and here we show that the epitope is in the region between amino acids 855 and 902 (the numbering of the H+/K(+)-ATPase catalytic subunit throughout the paper refers to the rabbit sequence). The localization of this epitope in conjunction with previously observed trypsin cleavage sites in the C-terminal one third of the enzyme and the hydrophobicity plot of the deduced peptide sequence are evidence for a structural model for the alpha-subunit of the H+/K(+)-ATPase which contains at least ten membrane spanning segments, similar to that deduced for the Ca(2+)-ATPase of sarcoplasmic reticulum.

The H+/K(+)-ATPase is the dimeric enzyme responsible for H+ secretion by the gastric parietal cells. The present study examined the response of rat fundic mRNA levels of H+/K(+)-ATPase alpha-subunit and somatostatin to the inhibition of H+/K(+)-ATPase enzyme activity and gastric pH elevation by oral omeprazole administration. Omeprazole inhibits the alpha-subunit of H+/K(+)-ATPase covalently and stabilizes stimulated morphology of the parietal cell. After a single administration of omeprazole (100 mg/kg), H+/K(+)-ATPase alpha-subunit mRNA levels increased significantly by 57% at 3 h and remained elevated for 6 h, returning to the basal level by 24 h. After multiple administrations of omeprazole (100 mg/kg per day, every 24 h for 3 days), H+/K(+)-ATPase alpha-subunit mRNA levels were already elevated at the time of the last dose, reached maximum at 6 h (95% increase above control), and returned to the pre-treatment level after 36 h. Nuclear run-on assay indicated H+/K(+)-ATPase gene transcription was significantly increased by omeprazole pretreatment in vivo. In contrast, a significant decrease in fundic somatostatin mRNA occurred at 12 h after a single dose, and the inhibition was more pronounced and lasted longer after multiple doses of omeprazole. These data indicate that omeprazole, while effectively inhibiting H+/K(+)-ATPase activity, induces H+/K(+)-ATPase gene expression in the parietal cells. An inverse relationship exists between the regulation of somatostatin gene expression in fundic D-cells and H+/K(+)-ATPase gene expression. The increase in H+/K(+)-ATPase alpha-subunit mRNA could be due to alterations in extracellular gastrin/somatostatin ratios or could be induced by intracellular effects of omeprazole.

The substituted benzimidazole, picoprazole, inhibited the gastric (H+ + K+)-ATPase in a concentration-and time-dependent manner. Half-maximal inhibition of the (H+ + K+)-ATPase activity was obtained at about 2 . 10(-6)M under standard conditions. In addition to the inhibition of ATPase activity, parallel inhibition of phosphoenzyme formation and the proton transport activity were achieved. Radiolabelled picoprazole was found to bind to 100 kDa peptide; this peptide was shown by phosphorylation experiments to contain the catalytic centre of the (H+ + K+)-ATPase. Studies on the (Na+ + K+)-ATPase indicated that this enzyme was unaffected by picoprazole. From the data presented and from other pharmacological studies, it is proposed that this compound inhibits acid secretion at the level of the parietal cell by its ability to inhibit the gastric proton pump, the (H+ + K+)-ATPase.

The photoaffinity ligand 8-azidoadenosine 3′,5-monophosphate was employed to label cAMP binding proteins in both fractionated and freeze-thawed rabbit gastric glands. Fractionated glands incorporated the azido-cAMP label primarily into two cytosolic proteins with apparent molecular weights of 58 000 and 48 000. No enrichment of label was found in fractions containing basolateral or apical membranes. DEAE-cellulose chromatography of the cytosolic proteins resulted in the separation of two cAMP-dependent protein kinase peaks. Azido cAMP labelling of each peak suggested the initial peak contained type I cAMP-dependent protein kinase while the second peak contained the type II kinase. Labelling of ‘resting’ gastric glands resulted in radioactive proteins of apparent molecular weights of 58 000.and 48 000. When gastric glands were stimulated to produce acid by the addition of 10 −4 M histamine or 1 mM dibutyryl cAMP there was 32–44% dimunition of ligand incorporation compared to control glands. The results strongly suggest that histamine- mediated stimulus-secretion coupling in gastric glands involves activation of parietal cell cAMP-dependent protein kinases.

Fluorescein isothiocyanate was used to covalently label the gastric (H+ + K+)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated p-nitrophenylphosphatase activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of pI 5.6 was also noted. Fluorescence was quenched by K+, Rb+ and Tl+ in a dose-dependent manner, and the K0.5 values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg2+-dependent fluorescent quench. Ca2+ reversed the K+-dependent loss of fluorescence and inhibited it when added prior to K+. This may relate to the slow phosphorylation in the presence of ATP found when Ca2+ was substituted for Mg2+ and the absence of K+-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K+ binding to the enzyme.

Lysosomes (tritosomes) were purified from the livers of rats injected with Triton WR 1339. The lysosomes developed an Mg2+-ATP-dependent pH gradient as measured by Acridine orange accumulation. H+ transport was supported by chloride, but not sulfate, and was independent of the cation used. H+ transport and Mg2+-stimulated ATPase was inhibited by diethylstilbesterol (K0.5 = 2 microM). N-Ethylmaleimide inhibited H+ transport (K0.5 = 30 microM). At low concentrations of N-ethylmaleimide, ATP partially protected H+ transport from inhibition with N-ethylmaleimide. Photolysis with 8-azido-ATP inhibited H+ transport and Mg2+-stimulated ATPase activity. Under these same conditions, 8-azido-[alpha-32P]ATP reacted with a number of polypeptides of the intact lysosome and lysosomal membranes. Pump-dependent potentials were measured using the fluorescent potential-sensitive dye, DiSC3(5) (3,3'-dipropylthiocarbocyanine) and ATP-dependent potential generation was inhibited by diethylstilbesterol. Chloride, but not sulfate reduced the magnitude of the ATP-dependent membrane potential, as measured using merocyanine 540. The chloride conductance, independent of ATP, was of sufficient magnitude to generate a H+ gradient driven by external chloride in the presence of tetrachlorosalicylanilide. In Cl- free media, ATP-dependent H+ transport was restored to control levels by outwardly directed K+ gradients in the presence of valinomycin. The role of cell Cl- is to provide the necessary conductance for supporting lysosomal acidification by the electrogenic proton pump.

Methods are described for purification of a vesicular membrane fraction of hog gastric mucosa using differential centrifugation, density gradient separation on zonal rotors and free-flow electrophoresis. As a result a fraction is obtained enriched 40-fold in terms of K+-ATPase and free of any other enzyme marker other than K+-activated p- nitrophenyl phosphatase. the 5′-nucleotidase and basal Mg2+-ATPase are clearly separated from the latter enzymes. Osmotic shock, Triton X-100 treatment or K+ ionophores increased the K+-ATPase activity in isotonic conditions, but K + -p- nitrophenyl phosphatase is not affected by these treatments, nor is the ATPase activity in the presence of NH4+. The results suggest that the electrophoretic fraction contains a major population of tight vesicles, whose permeability to K+ is rate limiting for the ATPase activity but not for the p- nitrophenyl phosphatase activity. It is concluded that K+ site for the ATPase is internal whereas the K+ site for the p- nitrophenyl phosphatase is external, hence, the K+ site must be mobile across the membrane.

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.

An ATPase has been solubilized from dog gastric mucosa which was localized to the smooth vesicular fraction of the tissue homogenate. With solubilization, there was a marked increase in the HCO 3 − activation of the enzyme, and several other oxyanions were found to stimulate ATPase activity, such as borate, selenite, arsenite, arsenate and sulfite. The stimulation was a function of the concentration and pK of the base. Additionally, methane sulfonyl chloride inhibited the enzyme, particularly the base activated component. Based on these observations a tentative model for the HCO 3 − activation mechanism is suggested.

An ATPase was isolated and partially purified from mammalian pancreas. It was shown to be stimulated several fold by HCO3− and other anions, and to be completely inhibited by SCN− and CNO−. In many of its properties it resembles gastric ATPase and a model is suggested to account for pancreatic HCO3− secretion.

Using zonal sucrose density gradient centrifugation methods to fractionate the subcellular components in gastric mucosal homogenates have been developed. Methods are described which give high yield or rapidity in fractionation. A procedure is described which enables large-scale preparations of smooth walled vesicular membranes containing HCO 3 − -ATPase activity free from mitochondrial contamination as assessed by electron microscopic morphology and undetectable succinic dehydrogenase or monoamine oxidase activity. A method to purify gastric mitochondria is also described.

When pig brain microsomes or NaI-treated microsomes are preincubated with ATP and NH 2 OH, phosphorylation from [γ- 32 P]ATP in the presence of Mg 2+ plus Na + is inhibited by 80% or more, whereas hydrolysis is only slightly reduced. This shows that NH 2 OH is capable of reacting with the native enzyme, and various explanations for these findings are discussed.

Acid rate, resistance, short circuit current, P.D., Cl− transport, O2 consumption and redox steady state were determined in frog or Necturus gastric mucosa under a variety of conditions. It was shown that measurements of ΔH/ΔO2 under conditions of stimulation of acid rate by hormones in Necturus, or ion substitution in frog did not allow valid conclusions to be drawn as to acid mechanism. The simplest interpretation of the results of a series of experiments using redox inhibitors and phosphorylative inhibitors and uncouplers is that mitochondrially generated ATP is the obligatory energy source for H+ transport, and that Cl− transport can utilize extramitochondrial sources of energy.