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G Sachs

Publications and source records attributed to G Sachs.

At least 55 records · Page 3Linked to original sources
PubMed2000

Review article: the control of gastric acid and Helicobacter pylori eradication.

This review focuses on the gastric acid pump as a therapeutic target for the control of acid secretion in peptic ulcer and gastro-oesophageal reflux disease. The mechanism of the proton pump inhibitors is discussed as well as their clinical use. The biology of Helicobacter pylori as a gastric denizen is then discussed, with special regard to its mechanisms of acid resistance. Here the properties of the products of the urease gene clusters, ureA, B and ureI, E, F, G and H are explored in order to explain the unique location of this pathogen. The dominant requirement for acid resistance is the presence of a proton gated urea transporter, UreI, which increases access of gastric juice urea to the intrabacterial urease 300-fold. This enables rapid and continuous buffering of the bacterial periplasm to approximately pH 6.0, allowing acid resistance and growth at acidic pH in the presence of 1 mM urea. A hypothesis for the basis of combination therapy for eradication is also presented.

Gastric Acid↗
PubMed2000

Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier.

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3',4', 5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB-kanR-EFGH operon all showed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited access of urea to internal urease at neutral pH. Measurement of 14C-urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.

Acids↗
PubMed2000

Role of PACAP1 receptor in regulation of ECL cells and gastric acid secretion by pituitary adenylate cyclase activating peptide.

We previously reported that PAC1 is expressed on ECL cells resulting in stimulation of [Ca2+]i, histamine and acid secretion. The study reported here characterized the signaling by PAC1 on ECL cells; determined the effects of PACAP on the gastric acid secretion in vivo, and determined the effects of chronic administration of PACAP-27 on ECL cell proliferation. PACAP-27 dose dependently stimulated ECL cell Ca2+ and AC with detectable stimulation at 1 nM and maximal stimulation at 100 nM (six-fold). In rats PACAP-27 administration (10 pmol/kg/h) increased the rate of gastric acid secretion when an antisomatostatin antibody was co-administered. Chronic administration of PACAP (10 pmol/h for seven days) via osmotic pump resulted in a more than twofold increase in BrdU incorporation into ECL cells. PACAP acting at the PAC1 results in dual signaling responses to both [Ca2+]i. AC in ECL cells stimulates gastric acid secretion via the actions of histamine acting at the parietal cell and in whole animals leads to proliferation of ECL cells when administered chronically.

Adenylyl Cyclases↗
PubMed2000

Expression of the Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease.

ureI encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of Helicobacter pylori and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we produced H. pylori ureI deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determined ureI expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastric Helicobacter species. ureI was found to be present in the genome of the gastric Helicobacter species and absent in the nongastric Helicobacter species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacter species. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacter species. The ureI gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

Bacterial Proteins↗
PubMed2000

Regulation of parietal cell calcium signaling in gastric glands.

The ligands interacting with enterochromaffin-like (ECL) and parietal cells and the signaling interactions between these cells were investigated in rabbit gastric glands using confocal microscopy. Intracellular calcium concentration ([Ca(2+)](i)) changes were used to monitor cellular responses. Histamine and carbachol increased [Ca(2+)](i) in parietal cells. Gastrin (1 nM) increased [Ca(2+)](i) in ECL cells and adjacent parietal cells. Only the increase of [Ca(2+)](i) in parietal cells was inhibited by H(2) receptor antagonists (H(2)RA). Gastrin (10 nM) evoked an H(2)RA-insensitive [Ca(2+)](i) increase in parietal cells. Carbachol produced large H(2)RA- and somatostatin-insensitive signals in parietal cells. Pituitary adenylate cyclase-activating peptide (PACAP, 100 nM) elevated [Ca(2+)](i) in ECL cells and adjacent parietal cells. H(2)RAs abolished the PACAP-stimulated [Ca(2+)](i) increase in adjacent parietal cells. Somatostatin did not inhibit the increase of [Ca(2+)](i) in parietal cells stimulated with histamine, high gastrin concentrations, or carbachol but abolished ECL cell calcium responses to gastrin or PACAP. Hence, rabbit parietal cells express histaminergic, muscarinic, and CCK-B receptors coupled to calcium signaling but insensitive to somatostatin, whereas rabbit and rat ECL cells express PACAP and CCK-B calcium coupled receptors sensitive to somatostatin.

Animals↗
PubMed2000

Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells.

Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease. We employed pH- and Ca(2+)-sensitive fluorescent dyes and confocal microscopy to determine the location, rate, and magnitude of pH changes in an H. pylori-AGS cell coculture model, comparing wild-type bacteria with nonpolar ureI-deletion strains (ureI-ve). Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial periplasmic pH, followed by an increase of medium pH and then pH in AGS cells. No change in periplasmic pH occurred in ureI-deletion mutants, which also induced a slower increase in the pH of the medium. Pretreatment of the mutant bacteria with the detergent C(12)E(8) before adding urea resulted in rapid elevation of bacterial cytoplasmic pH and medium pH. UreI-dependent NH(3) generation by intrabacterial urease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice. Perfusion of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or [Ca(2+)](in), unless the conditioned medium was first neutralized to elevate the NH(3)/NH(4)(+) ratio. Therefore, cellular effects of intrabacterial ammonia generation under acidic conditions are indirect and not through a type IV secretory complex. The pH(in) and [Ca(2+)](in) elevation that causes the NH(3)/NH(4)(+) ratio to increase after neutralization of infected gastric juice may contribute to the gastritis seen with H. pylori infection.

Ammonia↗
PubMed2000

Analysis of the membrane domain of the gastric H(+)/K(+)-ATPase.

A structure of the catalytic or alpha subunit of the H(+)/K(+)-ATPase, with ten transmembrane segments, and of the beta subunit, with a single such segment, was established using a combination of tryptic cleavage and peptide sequencing and in vitro translation. Sites at which covalent ligands bind to external surfaces were also defined by cleavage, separation and sequencing. Cys813 was found to be the common covalent binding site for all the substituted pyridyl methylsulfinyl benzimidazoles. The binding region of a K(+)-competitive reagent, the 1,2 &agr; -imidazo-pyridine SCH 28080, was defined by the kinetic effects of site-specific mutations. Amino acids substitutions in membrane-spanning segments M1, M3, M4 and M6 were found to influence the apparent inhibitor constant, K(i), to varying degrees, some having a large effect, some a moderate effect and some a slight effect, whereas some mutations had no effect. We interpret changes in K(i) without effects on the apparent Michaelis constant, K(m), as affecting SCH 28080 binding only. Mutation of Cys813 significantly affected the K(i) for SCH 28080, explaining the prevention of benzimidazole inhibition by the imidazo-pyridine.A model of the &agr; subunit was constructed with a vestibule on the luminal surface of the pump bounded by M1-M6 and containing the SCH 28080 binding region. The cation binding site is suggested to be more towards the cytoplasmic face of the enzyme's membrane domain. This model predicts the membrane peptide associations for the catalytic subunit. Biochemical and yeast two-hybrid methods place the beta subunit in association with M8, whereas similar methods place M5/6 in proximity to M9/10. These results, when combined with analysis of the two-dimensional crystals of the sarcoplasmic reticular Ca(2+) and Neurospora crassa H(+)-ATPases, provide the basis for a tentative model of the arrangement of the six core segments of the gastric H(+)/K(+)-ATPase.

Animals↗
PubMed2000

[Nitrous oxide free low-flow anesthesia].

The routine use of nitrous oxide as a component of the carrier gas has been unanimously called into question in recent surveys, in fact, its use is now recommended in indicated cases only. Whereas a lot of contraindications are listed in the surveys, precise definitions of justified indications are not given. In clinical routine practice, there are absolutely no problems in carrying out inhalational anaesthesia without nitrous oxide. The missing analgetic effect can be compensated for by moderately increasing the additively used amount of opioids, while the missing hypnotic effect can be achieved by raising the expired concentration of the inhalational anaesthetic by not more than 0.2-0.25 x MAC. Thus, when isoflurane is used, an expired concentration of 1.2 vol% is desired, in the case of sevoflurane of 2.2 vol% and with desflurane of 5.0 vol%. In addition, doing without nitrous oxide facilitates the performance of low flow anaesthetic techniques considerably. Since the patient only inhales oxygen and the volatile anaesthetic, the total gas uptake is reduced significantly. Washing out nitrogen is no longer necessary. This means that the initial phase of low flow anaesthesia, during which high fresh gas flows have to be used, can be kept short. Its duration is now determined by the wash-in of the volatile anaesthetic. Since there is no uptake of nitrous oxide, a considerably greater volume of gas is circulating within the breathing system, minimizing the possibility of accidental gas volume deficiency. Thus, if anaesthesia machines with highly gas-tight breathing systems are used, even the performance of non-quantitative closed system anaesthesia becomes possible in routine clinical practice. The carrier gas flow can be reduced to just that amount of oxygen which is really taken up by the patient. This oxygen volume can be roughly calculated by applying the Brody's formula. Using fresh gas flows as low as 0.25 l/min, however, will result in a significant decrease of the output of conventional vaporizers outside the circuit. Thus, it becomes nearly impossible to maintain an expired isoflurane concentration of 1.2 vol%. With respect to their pharmcokinetic properties, the newer low soluble volatile agents sevoflurane and desflurane are better suited for use with flows corresponding to the basal oxygen uptake. Our own clinical experience, gained in the last six months from a trial involving over 1,800 patients, shows that the increase in opioid consumption resulted in additional costs of about 0.25-0.50 DM per patient. The increased concentration of inhalational agents brought additional costs of 3.00 to 5.00 DM for a two-hour anaesthesia. On the other hand, doing without nitrous oxide saved 2.61 DM per one-hour anaesthesia, whereby our consumption of nitrous oxide is extremely low as minimal flow anaesthesia is performed consistently. Furthermore, these calculations disregard the cost of the technical maintenance fo the central gas piping system and of the regular measurement of workplace contamination with nitrous oxide by a certified institute, which in Germany, ad least, is obligatory. The additional costs of nitrous oxide-free inhalational anaesthesia seem to be balanced by the savings. Given the numerous justified arguments against the routine use of nitrous oxide, the lack of precisely-defined indications and the clinical experience showing that doing without nitrous oxide is uncomplicated, self-financing and ecologically beneficial, the use of nitrous oxide should be given up completely.

Anesthesia, Inhalation↗
PubMed2000

[Prevalence of psychiatric disorders in elderly inpatients of medical and surgical departments].

OBJECTIVE: The aim of the present survey was to study the psychiatric prevalence among elderly patients of medical and surgical wards in Austria. METHODS: 244 inpatients, 60 years and above, were investigated by research psychiatrists using the Clinical Interview Schedule. RESULTS: The prevalence was found to be 38.7% in medical departments, and 33.0% in surgical departments. According to DSM-III-R criteria, dementia (20.9%) and minor depression (7.0%) were the most frequent psychiatric categories, followed by substance abuse disorders (4.5%). Among those suffering from psychiatric disorders, 68.5% received psychotropics. Only a quarter of these drugs were prescribed by consulting psychiatrists, while about three quarters were ordered by ward physicians. CONCLUSIONS: These facts underline the importance of an intensive cooperation between specialists for somatic medicine and psychiatrists.

Aged↗
PubMed1999

Membrane insertion scanning of the human ileal sodium/bile acid co-transporter.

Mammalian sodium-dependent bile acid transporters (SBATs) responsible for bile salt uptake across the liver sinusoidal or ileal/renal brush border membrane have been identified and share approximately 35% amino acid sequence identity. Programs for prediction of topology and localization of transmembrane helices identify eight or nine hydrophobic regions for the SBAT sequences as membrane spanning. Analysis of N-linked glycosylation has provided evidence for an exoplasmic N-terminus and a cytoplasmic C-terminus, indicative of an odd number of transmembrane segments. To determine the membrane topography of the human ileal SBAT (HISBAT), an in vitro translation/translocation protocol was employed using three different fusion protein constructs. Individual HISBAT segments were analyzed for signal anchor or stop translocation (stop transfer) activity by insertion between a cytoplasmic anchor (HK M0) or a signal anchor segment (HK M1) and a glycosylation flag (HK beta). To examine consecutive HISBAT sequences, sequential hydrophobic sequences were inserted into the HK M0 vector or fusion vectors were made that included the glycosylated N-terminus of HISBAT, sequential hydrophobic sequences, and the glycosylation flag. Individual signal anchor (SA) and stop transfer (ST) properties were found for seven out of the nine predicted hydrophobic segments (H1, H2, H4, H5, H6, H7, and H9), supporting a seven transmembrane segment model. However, the H3 region was membrane inserted when translated in the context of the native HISBAT flanking sequences. Furthermore, results from translations of sequential constructs ending after H7 provided support for integration of H8. These data provide support for a SBAT transmembrane domain model with nine integrated segments with an exoplasmic N-terminus and a cytoplasmic C-terminus consistent with a recent predictive analysis of this transporter topology.

Amino Acid Sequence↗
PubMed1999

Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1.

Binding of vascular endothelial growth factor (VEGF) to its receptor, VEGFR-2 (Flk-1/KDR), induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for signaling proteins that relay the signals for cell proliferation, migration, and permeability enhancement. We explored the VEGF/receptor signaling pathway by performing a two-hybrid screen of a rat lung cDNA library in yeast using the intracellular domain of rat VEGFR-2 as bait. Two clones encoding lipocortin V were isolated. Subsequent studies with the yeast two-hybrid assay showed that the complete intracellular domain of VEGFR-2 was required for the interaction. Co-immunoprecipitation of translated proteins confirmed the interaction between the VEGF receptor and lipocortin V. VEGF induced a rapid tyrosine phosphorylation of lipocortin V in human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with antisense oligodeoxyribonucleotide (ODN) for lipocortin V significantly inhibited VEGF-induced cell proliferation, which was accompanied by a decrease in protein synthesis and tyrosine phosphorylation of lipocortin V. Our results indicate that lipocortin V may function as a signaling protein for VEGFR-2 by directly interacting with the intracellular domain of the receptor and appears to be involved in regulation of vascular endothelial cell proliferation mediated by VEGFR-2.

Animals↗
PubMed1999

Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for p2-type atpases.

The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na, K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

Animals↗
PubMed1999

beta-subunit assembly is essential for the correct packing and the stable membrane insertion of the H,K-ATPase alpha-subunit.

The alpha-subunits of H,K-ATPase (HKAalpha) and Na,K-ATPase require a beta-subunit for maturation. We investigated the role of the beta-subunit in the membrane insertion and stability of the HKAalpha expressed in Xenopus oocytes. Individual membrane segments M1, M2, M3, M4, and M9 linked to a glycosylation reporter act as signal anchor (SA) motifs, and M10 acts as a partial stop transfer motif. In combined HKAalpha constructs, M2 acts as an efficient stop transfer sequence, and M3 acts as a SA sequence. However, M5 and M9 have only partial SA function, and M7 has no SA function. Consistent with the membrane insertion properties of segments in combined alpha constructs, M1-3 alpha-proteins are resistant to cellular degradation, and M1-5 up to M1-10 alpha-proteins are not resistant to cellular degradation. However, co-expression with beta-subunits increases the membrane insertion of M9 in a M1-9 alpha-protein and completely protects M1-10 alpha-proteins against cellular degradation. Our results indicate that HKAalpha N-terminal (M1-M4) membrane insertion and stabilization are mediated by intrinsic molecular characteristics; however, the C-terminal (M5-M10) membrane insertion and thus the stabilization of the entire alpha-subunit depend on intramolecular and intermolecular beta-subunit interactions that are similar but not identical to data obtained for the Na,K-ATPase alpha-subunit.

Animals↗
PubMed1999

Role of neuropeptide-sensitive L-type Ca(2+) channels in histamine release in gastric enterochromaffin-like cells.

Peptides release histamine from enterochromaffin-like (ECL) cells because of elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) by either receptor-operated or voltage-dependent Ca(2+) channels (VDCC). To determine whether VDCCs contribute to histamine release stimulated by gastrin or pituitary adenylate cyclase-activating polypeptide (PACAP), the presence of VDCCs and their possible modulation by peptides was investigated in a 48-h cultured rat gastric cell population containing 85% ECL cells. Video imaging of fura 2-loaded cells was used to measure [Ca(2+)](i), and histamine was assayed by RIA. Cells were depolarized by increasing extracellular K(+) concentrations or by 20 mM tetraethylammonium (TEA(+)). Cell depolarization increased transient and steady-state [Ca(2+)](i) and resulted in histamine release, dependent on extracellular Ca(2+). These K(+)- or TEA(+)-dependent effects on histamine release from ECL cells were coupled to activation of parietal cells in intact rabbit gastric glands, and L-type channel blockade by 2 microM nifedipine inhibited 50% of [Ca(2+)](i) elevation and histamine release. N-type channel blockade by 1 microM omega-conotoxin GVIA inhibited 25% of [Ca(2+)](i) elevation and 14% of histamine release. Inhibition was additive. The effects of 20 mM TEA(+) were fully inhibited by 2 microM nifedipine. Both classes of Ca(2+) channels were found in ECL cells, but not in parietal cells, by RT-PCR. Nifedipine reduced PACAP-induced (but not gastrin-stimulated) Ca(2+) entry and histamine release by 40%. Somatostatin, peptide YY (PYY), and galanin dose dependently inhibited L-type Ca(2+) channels via a pertussis toxin-sensitive pathway. L-type VDCCs play a role in PACAP but not gastrin stimulation of histamine release from ECL cells, and the channel opening is inhibited by somatostatin, PYY, and galanin by interaction with a G(i) or G(o) protein.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

PACAP type I receptor activation regulates ECL cells and gastric acid secretion.

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in gastric nerves, and PACAP receptors (PAC1) are found on gastric enterochromaffin-like (ECL) cells. Expression of PAC1 splice variants in purified ECL cells was determined by RT-PCR. PACAP effects on ECL cells were analyzed by video imaging of [Ca(2+)](i) and histamine release; its effects on gastric glands were examined by confocal microscopy of [Ca(2+)](i) in ECL and parietal cells. PACAP action on D cells was measured by [Ca(2+)](i) and radioimmunoassay. PACAP effects on acid secretion were determined in fistula rats with or without neutralizing anti-somatostatin antibodies. All splice variants of PAC1 were found, but vasoactive intestinal polypeptide (VIP) receptor (VPAC) products were absent. PACAP-27 and -38 dose-dependently raise [Ca(2+)](i) in ECL cells, and stimulated histamine release. VIP had a much lower affinity, which demonstrates the presence of PAC1 but not VPAC. PACAP elevated [Ca(2+)](i) in ECL and parietal cells of superfused gastric glands, but only the parietal cell signal was inhibited by ranitidine, showing the absence of PAC1 on parietal cells, and demonstrating functional coupling between the cell types. PACAP and VIP stimulated calcium signaling and somatostatin release from D cells with almost equal efficacy. Acid secretion was stimulated after intravenous injection of PACAP into rats treated with somatostatin antibody. PACAP is a candidate as a mediator of neural regulation of acid secretion.

Alternative Splicing↗
PubMed1998

The pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1-R) is expressed on gastric ECL cells: evidence by immunocytochemistry and RT-PCR.

The current study was undertaken to determine the presence and distribution of PAC1-Rs within the gastric mucosa. Polyclonal antibodies to the carboxyl terminus of the rat PAC1-R were generated and shown to be specific against the PAC1-R expressed in NIH 3T3 cells. Western blot analysis using isolated (approximately 85% pure) ECL cell membranes identified a 48 kD protein consistent with the calculated molecular mass of the cloned PAC1-R. RT/PCR performed using specific primers for the PAC1-R confirmed the presence of splice variants of the rat PAC1-R, but not VPAC1-R or VPAC2-R. These data provide the first direct evidence for the existence of functional PACAP Type I receptors on ECL cells of the gastric mucosa and suggest a potential role for PACAP in the stimulation of gastric acid secretion and in the regulation of the growth of ECL cells.

3T3 Cells↗
PubMed1998

Identification of the site of inhibition by omeprazole of a alpha-beta fusion protein of the H,K-ATPase using site-directed mutagenesis.

The alpha subunit of eukaryotic P-type ATPases has ten experimentally defined transmembrane or membrane inserted segments. The fifth and sixth of these are short, not predicted by hydropathy analysis, do not insert independently into microsomal membranes, and are readily removed after tryptic digestion and therefore may be membrane inserted sequences. Acid transport by the gastric H, K-ATPase is covalently inhibited by several substituted pyridyl methylsulfinyl benzimidazoles, such as omeprazole. These act as probes of accessible extracytoplasmic thiols because they are accumulated in the acid transporting gastric vesicles and then convert to thiol reactive, cationic tetracyclic sulfenamides. Inhibition is due mainly to disulfide formation with Cys813 or Cys822 in M5/6 and perhaps with a contribution from Cys892 in the loop between transmembrane segment (TM) 7 and TM8. Identification of the specific cysteine responsible for inhibition should be able to define the turn between M5 and M6. The gastric H,K-ATPase alpha-beta heterodimer was expressed as a fusion protein in HEK 293 cells. Transient transfection resulted in most of the protein being retained in the endoplasmic reticulum with only core glycosylation and minor activity of the ATPase evident. Stable transfection resulted in plasma membrane localization of the protein and complex glycosylation. The transfected but not the control cells displayed cation-stimulated, SCH 28080-inhibited ATPase activity and SCH 28080- and omeprazole-inhibited 86Rb uptake. The two cysteines in M5/6 and Cys892 in the TM7/8 loop were mutated to the amino acids found in the Na,K-ATPase in order to determine which of the three cysteine residues were important for benzimidazole inhibition. Mutation of one, two, or all three cysteines did not alter enzyme activity, 86Rb transport, or SCH 28080 inhibition. Only removal of Cys822 blocked omeprazole inhibition of 86Rb transport. These data suggest that Cys822 is present in a region of the enzyme most easily accessed by the cationic sulfenamide formed by omeprazole, presumably the turn between M5 and M6.

Amino Acid Sequence↗