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J G Forte

Publications and source records attributed to J G Forte.

At least 37 records · Page 2Linked to original sources
PubMed1997

ME-3407, a new antiulcer agent, inhibits acid secretion by interfering with redistribution of H(+)-K(+)-ATPase.

ME-3407 is a newly developed antiulcer drug that markedly promoted the healing of acetic acid-induced chronic ulcers in rats presumably due to potent inhibition of acid secretion. ME-3407 and its metabolites, the sulfoxide of which was preserved, produced dosedependent inhibition of aminopyrine accumulation by rabbit gastric glands stimulated by any agonist, suggesting that the site of their action was downstream from the production of second messengers. Although one of the metabolites, EF-4025, showed some inhibitory effects on functional activities of H(+)-K(+)-ATPase, ME-3407 itself was not a proton pump inhibitor. ME-3407, but not omeprazole, inhibited the stimulation-associated redistribution of H(+)-K(+)-ATPase from microsomes into the apical membranes in addition to delocalizing ezrin, a putative F-actin-membrane linker, from apical plasma membrane. ME-3407 and EF-4025 inhibited myosin light chain kinase (MLCK) and protein kinase A activities. Because another MLCK inhibitor, wortmannin, showed the same properties as ME-3407, i.e., inhibition of aminopyrine accumulation, inhibition of stimulation-associated redistribution of H(+)-K(+)-ATPase, and abnormal distribution of ezrin, we hypothesize that MLCK is one of the potential targets for the drug. We conclude that ME-3407 is a promising drug for treating peptic ulcers, as well as a useful tool for studying mechanisms of parietal cell activation, especially related to the recruitment and recycling of the proton pump.

Acetic Acid↗
PubMed1996

Biochemical characterization of ezrin-actin interaction.

The highly related actin isoforms are thought to have different functions. We recently demonstrated a polarized distribution of actin isoforms in gastric parietal cells and association of gastric ezrin with the cytoplasmic beta-actin isoform (Yao, X., Chaponnier, C., Gabbiani, G., and Forte, J. G. (1995) Mol. Biol. Cell. 6, 541-557). Here we used ultrastructural immunocytochemistry to verify that beta-actin is located within canalicular microvilli and the apical cortex of parietal cells, similar to the localization reported for ezrin. Furthermore, we tested whether ezrin binds preferentially to cytoplasmic beta-actin compared with the skeletal muscle alpha-actin isoform. Purified cytoplasmic beta-actin (from erythrocytes) and skeletal alpha-actin were assembled with gastric ezrin. Co-sedimentation experiments showed that gastric ezrin selectively co-pelleted with the beta-actin isoform and only very poorly with alpha-actin. Binding of erythrocytic beta-actin to ezrin is saturable with a molar ratio of approximately 1:10 (ezrin:actin) and a dissociation constant approximately 4.6 x 10(-8) M. In addition, ezrin promoted pyrene-labeled actin assembly, with predominant effects on filament elongation and a distinct preference for beta-actin compared with alpha-actin. Given these isoform-selective associations, we speculate that actin isoforms might segregate into different functional domains and exert specificity by interacting with isoform-orientated binding proteins.

Actins↗
PubMed1996

The Beta-subunit of the rabbit H,K-ATPase:a glycoprotein with all terminal lactosamine units capped with alpha-linked galactose residues.

The beta-subunit of the gastric H,K-ATPase is the most abundant glycoprotein in the tubulovesicular compartment of the acid-secreting parietal cells. The oligosaccharides of the beta-subunit have been shown to contain fucose, N-acetylglucosamine, mannose, galactose, and N-acetylgalactosamine. Previous studies have shown that the rabbit beta-subunit is devoid of N-acetylneuraminic acid. Here we report the structural features of the N-linked oligosaccharides of the beta-subunit from rabbit H,K-ATPase. We used glycosidase digestions and analysis by high-pH anion-exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization mass spectrometry to analyze the peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase F)- and endo-beta-N-acetylglucosaminidase H (Endo H)-released oligosaccharides. The studies showed that the oligosaccharides of the beta-subunit are a mixture of both oligomannosidic and lactosamine-type structures. The high-mannose structures were identified as Man5Man8GlcNAc2 species. A striking finding was that all the branches of the lactosamine-type structures were terminated with Galalpha-->Galbeta-->GlcNAc extensions. All of the lactosamine-type structures were found to be core fucosylated and some of them contained one to three lactosamine repeats. We propose that a part of the adaptation of the gastric beta-subunit to the acidic environment of the stomach is through providing acid-stable terminal residues on the oligosaccharides.

Amidohydrolases↗
PubMed1996

The membrane-recruitment-and-recycling hypothesis of gastric HCl secretion.

In the unstimulated oxyntic (or parietal) cell, the primary pump for gastric HCl secretion, the H+/K+-ATPase, is retained within the cytoplasm in a membranous compartment of tubulovesicles. Neural or hormonal stimulation of acid secretion induces extensive membrane transformations consistent with a fusion and recruitment of tubulovesicles to the apical plasma membrane. The consequent placement of H+/K+-ATPase in parallel with K(+) and Cl(-) channels provides the necessary ionic flow and ATP-driven exchange for net HCl secretion. Current evidence is consistent with a recruitment and recycling of membrane transporters, such as H+/K+-ATPase, through docking/fusion machinery analogous to that in many other systems.

Journal Article↗
PubMed1996

Exoglycosidase purity and linkage specificity: assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection.

Simplified HPLC protocols to determine the activity and linkage specificity and to detect the most commonly-encountered contaminants in available exoglycosidase preparations (Jacob and Scudder, Methods Enzymol., 230, 280-300, 1994) were developed. Monosaccharides and oligosaccharides were analyzed in a single chromatographic step using high-pH anion-exchange chromatography with pulsed amperometric detection. All analyses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the chromatograph. The sialidase from Newcastle disease virus was found to release both alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac from a triantennary, lactosamine-type oligosaccharide. The activity of alpha-galactosidase from green coffee beans was assayed using Gal alpha(1-->3)[Fuc-alpha(1ar2)]Gal by detection of Gal and Fuc alpha(1-->3)Gal. The linkage specificities of beta-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Gal beta(1-->3 or 4)GlcNAc beta(1-->3)beta(1-->4)Glc as substrates. Contaminating beta-N-acetylhexosaminidase activity in the beta-galactosidase preparation was assayed using an agalactobiantennary oligosaccharide. The alpha(1-->3 or 4) linkage specificity of fucosidase III from almond meal was confirmed (Scudder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked alpha(1-->6). An alpha-fucosidase from chicken liver was found to cleave alpha(1-->2,3 or 6)-linked Fuc residues from oligosaccharides. The activity of jack bean (Canavalia ensiformis) alpha-mannosidase was assayed with a relatively resistant substrate, Man alpha(1-->3)- Man beta(1-->4)GlcNAc. A GlcNAc beta(1-->4)-terminated triantennary oligosaccharide was used to assay for contaminating beta-N-acetylhexosaminidase activity in alpha-mannosidase preparations and to determine the linkage and branch specificity of beta-N-acetylhexosaminidase at different enzyme concentrations.

Animals↗
PubMed1996

Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity.

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.

Animals↗
PubMed1996

Stimulation of gastric acid secretion by cAMP in a novel alpha-toxin-permeabilized gland model.

It is generally believed that histamine-stimulated gastric acid secretion involves a transient elevation of intracellular Ca2+ and the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) cascade through phosphorylation, whose actions ultimately effect the fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing vesicles to the apical plasma membrane of parietal cells. To dissect the signaling events underlying gastric acid secretion, we have developed a permeabilized gastric gland model using Staphylococcus alpha-toxin. The advantage of this model is its ability to retain cytosolic components that are required for the secretory machinery. Here we show that acid secretion in alpha-toxin-permeabilized glands is a cAMP-dependent process, reaching a maximal stimulation at 100 microM cAMP. The cAMP-elicited acid secretion, as monitored by the accumulation of the weak base aminopyrine (AP), required functional mitochondria or exogenously supplied ATP. Maximal stimulation elicited by cAMP for AP uptake by permeabilized glands was 51-85% of intact glands. Moreover, secretory activity was potentiated by 0.1 mM ATP. The recruitment of H(+)-K(+)-ATPase-rich tubulovesicles into the apical plasma membrane was measured using biochemical and morphological assays, thus validating the cell activation processes in response to cAMP. From this permeabilized model, [gamma-32P]ATP was used to directly phosphorylate target proteins. A number of proteins whose phosphorylation-dephosphorylation is specifically modulated by cAMP were found. These studies establish the first permeabilized gland model in which the resting-to-secreting transition can be triggered and show that cAMP-mediated phosphorylation is correlated with secretory activity.

Aminopyrine↗
PubMed1995

Structural interactions between alpha- and beta-subunits of the gastric H,K-ATPase.

Structural and functional interactions between alpha- and beta-subunits of the H,K-ATPase were explored. The sensitivity to trypsinolysis of alpha-subunit was monitored by SDS-PAGE in control H,K-ATPase-enriched microsomes and in microsomes in which disulfide bonds of the beta-subunit were reduced using 2-mercaptoethanol (2-ME). Reduction of beta-subunit disulfide bonds increased the susceptibility of the alpha-subunit to tryptic digestion. Kinetics of trypsinolysis were also carried out in the presence of ligands known to bind with H,K-ATPase and favor a particular conformer state in the native enzyme. The time-course for release of tryptic peptides was monitored in protein stained gels and Western blots probed with monoclonal antibody alpha-H,K,12.18. In control preparations, where beta-subunit disulfides remained intact, trypsinolysis in the presence of ATP or K+ produced distinctive patterns of tryptic fragments, each characteristic of the conformational states induced by the respective ligand. For 2-ME-treated microsomes the altered alpha-subunit was unable to undergo ligand-induced conformational changes. The increased susceptibility of the alpha-subunit to trypsinization, the change in accessibility of tryptic cleavage sites and the inability of the alpha-subunit to undergo ligand-induced conformational changes after reduction of the beta-subunit disulfides suggest that the interactions between alpha- and beta-subunits are important for the conformational stability of the functional holoenzyme. A model localizing the most susceptible tryptic cleavage sites in control and 2-ME-reduced states is presented.

Animals↗
PubMed1995

Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.

Actins↗
PubMed1995

In vivo trafficking of nascent H(+)-K(+)-ATPase in rabbit parietal cells.

Protein metabolic labeling in vivo was used to determine a time course for trafficking of nascent H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) from endoplasmic reticulum (ER) to mature tubulovesicles in parietal cells. Stomachs of cimetidine-treated rabbits were taken 15-90 min after injection of [35S]methionine/cysteine, and mucosal microsomes were fractionated on sucrose gradients for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and autoradiography. After 15 min, labeled alpha-subunit peaked at approximately 1.14 g/ml, matching the distribution of the high-mannose beta-subunit precursor, "pre-beta." After 30 min, most labeled alpha-subunit was in a peak at approximately 1.10 g/ml, considered to be Golgi. By 90 min, most labeled alpha-subunit was in a light peak, at approximately 1.07 g/ml, aligned with the major peak of total H(+)-K(+)-ATPase previously characterized as mature tubulovesicles. From material enriched in pre-beta, alpha-subunit was coprecipitated with pre-beta by a terminal mannose-specific lectin, Galanthus nivalis agglutinin, in the same ratio as the mature alpha:beta ratio. Thus alpha- and beta-subunits associated early in the ER. This is the first use of protein metabolic labeling to study early trafficking of the H(+)-K(+)-ATPase in vivo. The techniques may be usefully applied to examining changes in H(+)-K(+)-ATPase synthetic rate in response to various pharmacological treatments and studying the divergent pathways for nascent H(+)-K(+)- and Na(+)-K(+)-ATPases.

Animals↗
PubMed1995

Functional significance of the beta-subunit for heterodimeric P-type ATPases.

We have reviewed the structural and functional role of the beta-subunit in a subfamily of the P-ATPases known as the alpha/beta-heterodimeric, cation-exchange ATPases. The subfamily consists of the various isoforms of Na+/K(+)-ATPase and H+/K(+)-ATPase, both of which pump a cation out of the cell (Na+ or H+, respectively) in recycle exchange for K+. Much of the earlier work has emphasized the functional activities of the alpha-subunit, which shares many characteristics with the broader P-ATPase family. It is now clear that the glycosylated beta-subunit is an essential component of the cation-exchange ATPase subfamily. All beta-subunit isoforms have three highly conserved disulfide bonds within the extracellular domain that serve to stabilize the alpha-subunit, alpha/beta interaction and functional activity of the holoenzyme. Evidence strongly suggests that the beta-subunit is involved in the K(+)-dependent reactions of the enzymes, such as the E1-E2 transition and K+ occlusion, and that the extracellular domain of the beta-subunit plays an important role in determining the kinetics of K+ interaction. In most vertebrate cells, the unassociated alpha-subunit is restricted to the endoplasmic reticulum (ER), and assembly of the alpha/beta complex occurs within the ER. Signals for exiting the ER and directing the correct intracellular trafficking are primarily determined by the beta-subunit; Na+/K(+)-ATPase typically terminates in the plasma membrane facing the basolateral membrane, whereas all isoforms of H+/K(+)-ATPase terminate in the apical membrane. The C-terminal extracellular domain of the beta-subunit is important for proper interaction with the alpha-subunit and for correct intracellular trafficking. Oligosaccharides on the beta-subunit are not essential for enzyme function, but do serve to enhance the efficiency of alpha/beta association by increasing the lifetime of the unassociated beta-subunit and the stability of the alpha/beta complex to tryptic attack. We propose that highly specialized glycosylation on the beta-subunit of the gastric H+/K(+)-ATPase may help to protect that enzyme from the harsh extracellular environment of the stomach.

Adenosine Triphosphatases↗
PubMed1994

Direct measurement of extracellular proton flux from isolated gastric glands.

We used the microphysiometer, a sensitive extracellular pH sensor, to resolve luminal (or apical) H+ secretion and basolateral release of OH- as well as liberation of acidic metabolites in rabbit gastric glands. Stimulation of glands via the adenosine 3',5'-cyclic monophosphate pathway produced a biphasic change in the extracellular acidification rate (EAR): after an initial transient decrease below the unstimulated baseline (-40.9 +/- 3.4%), the EAR increased to a steady-state maximal plateau (+98.1 +/- 5.3%) within 30 min (n = 37). We interpret the biphasic EAR profile as an initial excess of basolaterally released OH- followed by delayed luminal efflux of simultaneously produced H+. The elevated EAR at steady state reflected liberation of metabolic acid attributed to H(+)-K(+)-ATPase enzymatic activity. The presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid prevented OH- release and reduced steady-state EAR. Basolateral OH- release and steady-state EAR were also inhibited by the H(+)-K(+)-ATPase inactivators omeprazole and SCH-28080. Inhibition of Na+/H+ exchange did not reduce steady-state EAR and did not affect apical H+ production, as judged by the accumulation of the weak base aminopyrine. Sodium thiocyanate (1 mM), which short circuits intraluminal H+ accumulation, blocked OH- release, demonstrating its dependence on H(+)-OH- separation at the apical membrane. A computerized model was developed to illustrate how the observed biphasic EAR profile would result from a delayed luminal efflux of H+ due to transitory intraluminal compartmentalization.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Inhibiting gastric H(+)-K(+)-ATPase activity by omeprazole promotes degeneration and production of parietal cells.

Parietal cell morphology was studied after chronic inhibition of gastric H(+)-K(+)-adenosinetriphosphatase (ATPase) by omeprazole. Gastric mucosa from rabbits treated with omeprazole every 12 h (1 mg/kg sc) for 5 days were compared with sham-injected animals using immunohistochemistry and electron microscopy. Three immunocytochemical markers, including antibodies against the alpha- and beta-subunits of the H(+)-K(+)-ATPase, showed that some parietal cells in omeprazole-treated rabbits displayed light areas and granularity in their cytoplasm. These abnormalities were also apparent in semithin sections. Electron microscopy was used to categorize and quantitate the specific structural abnormalities in parietal cells. Cells were classified as normal, altered, or degenerated. For control tissues, altered and degenerated parietal cells were few; they collectively represented 6% of total parietal cells and were located mainly deep in the gland base. For omeprazole-treated tissues, altered and degenerated parietal cells occurred throughout the gland and averaged 62% of total parietal cells. In addition, macrophages invaded the mucosa presumably to eliminate degenerated cells. Although there was an increase in parietal cell degeneration, enhanced parietal cell generation was suggested by increases in mitosis among proliferative cells and, more specifically, in the number of preparietal cells. After 3 days of recovery from the omeprazole regimen, parietal cells and the gastric mucosa appeared to recover the normal morphology. In conclusion, blocking H(+)-K(+)-ATPase by omeprazole enhances degeneration and macrophage-mediated elimination of parietal cells and also causes an increase in preparietal cell production. Thus omeprazole temporarily changes the dynamic features of parietal cells in the rabbit to make them die early and grow fast.

Animals↗
PubMed1993

Monosaccharide and oligosaccharide analysis of proteins transferred to polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We have developed an intermediate method toward the complete carbohydrate analysis of proteins, which should be universally applicable to all proteins and independent of sample matrix. Using only Coomassie Blue-stained proteins which have been electroblotted onto polyvinylidene fluoride membranes, we report a strategy for: (i) determining unequivocally whether a protein is glycosylated; (ii) obtaining a complete monosaccharide composition; (iii) oligosaccharide mapping which separates most forms according to size, charge and isomerity; and (iv) sequentially releasing and analyzing specific classes of oligosaccharides with endoglycosidases. The method was shown to be applicable to a variety of well characterized soluble glycoproteins and to the membrane-bound protein, the gastric H+, K(+)-ATPase. The monosaccharide composition of the H+,K(+)-ATPase revealed the absence of N-acetylneuraminic or N-glycolylneuraminic acids and a monosaccharide composition which indicated O-linked sugar chains. Oligomannosidic/hybrid and biantennary oligosaccharides were sequentially released and analyzed from one electroblotted band of recombinant tissue plasminogen activator using endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F2, respectively. Sialylated polylactosamine structures were identified and quantified by analyzing high performance liquid chromatography profiles of oligosaccharides first released by peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase and then treated with endo-beta-galactosidase, using a single, stained band of recombinant erythropoietin. This recombinant erythropoietin was found to contain eight times more tetrasialylated oligosaccharides than previously reported (Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076); 47% of released oligosaccharides were identified as polylactosamine structures.

Animals↗
PubMed1993

Ezrin-calpain I interactions in gastric parietal cells.

Gastric ezrin, a membrane-cytoskeletal linker with sequence homology to talin and erythrocyte band 4.1, has been associated with the remodeling of parietal cell apical membrane that occurs with adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase stimulation. Here we examine the interrelationship between parietal cell ezrin and Ca(2+)-dependent protease activity. Addition of Ca2+ to sonicated gastric gland preparations rendered a relatively selective proteolysis of the 80-kDa ezrin, accompanied by the appearance of a 55-kDa breakdown product. Ca(2+)-dependent proteolysis of ezrin was blocked by E64, a cysteine protease inhibitor, or calpastatin, indicating calpain as the responsible protease. Degradation of ezrin in intact gastric glands was achieved by varying extracellular [Ca2+] and [ionomycin]. Ezrin degradation in situ was rapid and relatively selective, although Ca(2+)-dependent degradation of some spectrin-like bands was also observed. The effect of activated calpain I on parietal cell function was assessed by probing the secretory response to histamine stimulation using [14C]aminopyrine uptake, along with parallel measurements of calpain activity, over a wide range of ionomycin. Activation of calpain, as evidenced by loss of parietal cell ezrin, was correlated with decreased AP uptake by stimulated gastric glands, supporting a role for ezrin in the oxyntic secretory process. The calpain-ezrin interaction established here, and the similarities of calpain with talin and erythrocyte band 4.1, suggest a common feature to this family of ezrin/band 4.1/talin proteins that have been implicated in membrane-cytoskeletal association.

Animals↗
PubMed1993

Characterization of the beta-subunit of the H(+)-K(+)-ATPase using an inhibitory monoclonal antibody.

The gastric proton pump, H(+)-K(+)-ATPase, is composed of alpha- and beta-subunits. The 95-kDa alpha-subunit has been referred to as the catalytic subunit containing sites for ATP binding and phosphorylation. The beta-subunit is a glycoprotein with a 34-kDa core peptide that has a single transmembrane segment, a small cytoplasmic NH2-terminal, and a large extracellular COOH-terminal domain with seven potential N-linked glycosylation sites. To further study the beta-subunit, we developed monoclonal antibodies that identified a 52-kDa mannose-rich glycoprotein that was deglycosylated by endoglycosidase H such that six transient intermediates were identified, as well as a 34-kDa beta-subunit core peptide. These observations suggest that the beta-subunit is synthesized as a 52-kDa glycoprotein with seven N-linked precursor high-mannose oligosaccharides that mature into complex oligosaccharides. One antibody, 2G11, inhibits the K(+)-stimulated ATP hydrolysis as well as K(+)-stimulated p-nitrophenyl phosphatase (pNPPase) activity of the H(+)-K(+)-ATPase. Kinetic studies revealed that 2G11 inhibited maximum velocity (Vmax) of ATP hydrolysis by approximately 50% with no change in the Km for K+, whereas, for pNPPase both Vmax and Km were altered. These studies demonstrate a functional role for the beta-subunit in the H(+)-K(+)-ATPase activity, especially the K(+)-induced conformational states.

4-Nitrophenylphosphatase↗
PubMed1993

Omeprazole decreases H(+)-K(+)-ATPase protein and increases permeability of oxyntic secretory membranes in rabbits.

Amounts and fractional distributions of gastric H(+)-K(+)-adenosinetriphosphatase (ATPase) activity and H(+)-K(+)-ATPase protein as well as properties of H(+)-K(+)-ATPase-containing membranes were studied in rabbits injected with omeprazole (OM; 1 mg/kg sc twice daily for 5 days). Total H(+)-K(+)-ATPase activity decreased to 22 +/- 2% of control (n = 4). Densitometry of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots showed H(+)-K(+)-ATPase protein was decreased to 60-70% of control. In vitro reduction of the enzyme-OM disulfide bond with 0.1 M 2-mercaptoethanol increased microsomal H(+)-K(+)-ATPase activity to 56 +/- 7% of control (n = 3), consistent with a substantial decrease in enzyme protein. Incorporation of 35S-labeled methionine for 30 min before death resulted in 2.2-fold more label per unit of microsomal alpha-subunit protein (5 days OM vs. control). Thus the decrease in enzyme protein resulted from increased breakdown rather than decreased synthesis. A striking shift in distribution of H(+)-K(+)-ATPase-containing microsomes (tubulovesicles) on sucrose gradients reflected slow equilibration of most control vesicles with the gradient medium and faster equilibration after 5 days OM, indicating increased permeability. After 5 days OM, microsomal vesicle acidification (by acridine orange uptake assay) was negligible, even with 2-mercaptoethanol treatment, and H+ leakage on sudden delta pH was faster than control. We conclude that extended OM treatment not only inhibits H(+)-K(+)-ATPase but accelerates its breakdown and renders H(+)-K(+)-ATPase-containing membranes more permeable. It is thus possible that increased backward H+ flux contributes to profound inhibition of acid secretion during extended omeprazole treatment. In parallel experiments, H(+)-K(+)-ATPase activity and density gradient sedimentation of tubulovesicles returned to near normal 3 days after OM withdrawal.

Acridine Orange↗