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E Rabon

Publications and source records attributed to E Rabon.

17 recordsLinked to original sources
PubMed1992

Chemomechanical coupling in the gastric H,K ATPase.

The gastric H,K ATPase is investigated in terms of its secondary structure by analysis of the binding sites of the extracytoplasmic inhibitors and by tryptic cleavage of intact, inside-out gastric vesicles. The inhibitors affect phosphorylation and other partial reactions of the ATPase that depend on cytoplasmic conformational changes. The K competitive imidazopyridine, SCH28080 binds to the first pair of transmembrane segments, M1/M2, probably at phe124 and asp136. Omeprazole which generates a cationic sulfenamide in acid spaces binds to either cys813 or cys822 at one site and cys892 at the other. These cysteines are located at the membrane spanning pairs, M5/M6 and at M7/M8. Tryptic cleavage of intact inside out vesicles followed by labelling with fluorescein-5-maleimide provides direct evidence for 8 membrane spanning segments between positions 104/162 (M1/M2), 291/358(M3/M4), 776/835 (M5/M6),853/946 (M7/M8). Evidence is lacking so far for M9/M10, postulated on the basis of hydrophobicity for the Ca ATPase. Conformational studies suggest that there is interaction between the cytoplasmic loop between M4 and M5 (ATP domain) and the extracytoplasmic domain of the enzyme at the inhibitor binding sites.

Adenosine Triphosphate↗
PubMed1991

A K(+)-competitive fluorescent inhibitor of the H,K-ATPase.

The interactions of a novel fluorescent compound, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ) with the gastric H,K-ATPase were determined. MDPQ was shown to inhibit the H,K-ATPase and its associated K(+)-phosphatase competitively with K+, with Ki values of 0.22 and 0.65 microM, respectively. It also inhibited H+ transport with an IC50 of 0.29 microM, but at a concentration of 3.5 microM, reduced the steady-state level of phosphoenzyme by only 28%. The fluorescence of the inhibitor increased upon binding to the enzyme. 70% of this increment was quenched by K+, independently of Mg2+. The binding of MgATP to a high affinity site (K0.5(ATP) less than 1 microM) markedly increased the fluorescence due to the formation of an inhibitor-phosphoenzyme complex saturating with a K0.5(MDPQ) of 0.94 microM. The K(+)-dependent fluorescent quench (K0.5(K+) = 1.8 mM) required the ionophore, nigericin, indicating that K+ and MDPQ were competing at an extracytosolic site on the enzyme. Formation also of an enzyme-vanadyl-inhibitor complex was shown by the fact that Mg2+ plus vanadate enhanced MDPQ fluorescence in the absence of MgATP and decreased fluorescence in the presence of MgATP. The minimal stoichiometry of bound MDPQ determined by fluorescence titrations in the presence of MgATP was 1.4 mol/mol phosphoenzyme. The data suggest that this compound can serve as a probe of conformation at an extracytosolic site of the H,K-ATPase.

Adenosine Triphosphatases↗
PubMed1991

Identification of H+/K(+)-ATPase alpha,beta-heterodimers.

Glutaraldehyde treatment of the C12E8 solubilized H+/K(+)-ATPase crosslinks the catalytic subunit with an apparent molecular mass of 94 kDa in SDS polyacrylamide gels into two Coomassie stained particles migrating at approx. 147 and 173 kDa. The subunit composition of these particles was determined from the comparative distribution of FITC fluorescence, wheat germ agglutinin and anti-beta antibody reactivity in control and crosslinked preparations. FITC exclusively labelled the catalytic monomer of the native preparation and its fluorescence was initially distributed into two broad bands centered at approx. 147 and 173 kDa after crosslinking. These fluorescent bands coincided with the Coomassie stained particles. A glycoprotein(s) detected by wheat germ agglutinin reactivity was present in diffuse areas between 65 and 86 kDa and 95 to 134 kDa in the control preparation. This area was also labelled by the anti-beta antibodies. With crosslinking, the distribution of the wheat germ agglutinin reactive protein and anti-beta antibodies coincided with the crosslinked particles labelled by FITC. The presence of both the catalytic monomer and the beta subunit glycoprotein in the crosslinked particles indicated that these proteins were closely associated in the C12E8 solution. This suggests that the minimal structural particle of the H+/K(+)-ATPase is an alpha,beta-heterodimer.

Adenosine Triphosphatases↗
PubMed1988

SCH28080 prevents omeprazole inhibition of the gastric H+/K+-ATPase.

The interaction between SCH28080 and omeprazole, two specific inhibitors of gastric H+/K+-ATPase, was investigated using gastric glands and isolated gastric membranes. For gastric glands, inhibition of acid formation by SCH28080 was not reversed by washing whereas inhibition by omeprazole was partially reversed after washing. These features are opposite to what is found with isolated membranes. However, if gastric glands were permeabilized with digitonin after exposure to the inhibitors and recovery measured as ATP-dependent acid formation or H+/K+-ATPase activity, inhibition by SCH28080 was completely reversed while inhibition by omeprazole was non-reversible. Using a procedure of pretreatment with inhibitors followed by permeabilization and assay of recovered activity, it was found that a combined treatment with SCH28080 plus omeprazole prevented the irreversible inhibition by omeprazole, i.e. acid forming capability and ATPase activity were fully recovered. In order to test the possibility that SCH28080 prevented activation of omeprazole by dissipating an acid environment, control experiments were performed with SCN, which gave equivalent dissipation of the acid gradient but did not prevent the irreversible inhibition by omeprazole. These results were confirmed in isolated gastric membranes where residual p-nitrophenylphosphatase activity was assayed following exposure of acid transporting vesicles to omeprazole. Compared to control conditions, omeprazole inhibited 48% of the phosphatase activity whereas simultaneous addition of SCH28080 reduced the inhibition to 14%. The results therefore suggest that SCH28080 selectively blocks irreversible inhibition by omeprazole and thus that these two agents interact at a common region of the luminal aspect of the gastric H+/K+-ATPase.

Adenosine Triphosphatases↗
PubMed1987

Inhibition of gastric H+,K+-ATPase and acid secretion by SCH 28080, a substituted pyridyl(1,2a)imidazole.

A hydrophobic amine, SCH 28080, 2-methyl-8-(phenylmethoxy)imidazo(1,2a)pyridine-3-acetonitrile, previously shown to inhibit gastric acid secretion in vivo and in vitro, was also shown to inhibit basal and stimulated aminopyrine accumulation in isolated gastric glands when histamine, high K+ concentrations, or dibutyryl cAMP were used as secretagogues. Stimulated, but not basal, oxygen consumption was also inhibited. Neutralization of the acid space of the parietal cell by high concentrations of the weak base, imidazole, reduced the potency of the drug, suggesting that SCH 28080 was active when protonated. Studies on the isolated H+,K+-ATPase showed that the compound inhibited the enzyme competitively with K+, whether ATP or p-nitrophenyl phosphate were used as substrates. In contrast, the inhibition was mixed with respect to p-nitrophenyl phosphate and uncompetitive with respect to ATP. The drug reduced the steady state level of the phosphoenzyme but not the observed rate constant for phosphoenzyme formation in the absence of K+ nor the quantity of phosphoenzyme reacting with K+. The drug quenched the fluorescence of fluorescein isothiocyanate-modified enzyme and also inhibited the ATP-independent K+ exchange reaction of the H+,K+-ATPase. Its action on gastric acid secretion can be explained by inhibition of the H+,K+-ATPase by reversible complexation of the enzyme. This class of compound, therefore, acts as a reversible inhibitor of gastric acid secretion.

Adenosine Triphosphatases↗
PubMed1986

Crystallization of the gastric H,K-ATPase.

Crystalline arrays of the gastric H,K-ATPase were obtained in membrane preparations from hog and rabbit gastric mucosa. The lattice was formed rapidly in a medium containing K+, vanadate, Mg2+, and dimethyl sulfoxide at pH 6.0-6.9 in imidazole buffer from 4 to 22 degrees C. The crystal lattice exhibited P2 symmetry, and the unit cell dimension (a = 5.6, b = 11, and c = 10 nm) could accommodate 2 polypeptides of mass 116-129 kDa. In addition, the isolated preparation contained previously undescribed long cylindrical structures 16 nm thick. These structures consisted of a central core 6-7 nm wide from which particles spaced 5.5 nm apart protruded symmetrically.

Adenosine Triphosphatases↗
PubMed1985

Solubilization and reconstitution of the gastric H,K-ATPase.

Proteoliposomes containing the hog gastric H+,K+-ATPase were prepared from cholate and n-octyl glucoside extracts of native microsomes. Experiments were presented which show reconstitution-dependent selective purification of a 94-kDa peptide capable of Rb+/Rb+ exchange and active H+ transport. The absence of selective enrichment of residual protein contamination in this material suggests but does not prove that those transport reactions are attributable only to the 94-kDa peptide. Transport demonstrated inhibitor sensitivity and cation specificity comparable to the microsomal gastric ATPase. In K2SO4 media the H+ transport reaction was protonophore insensitive and correlated with MgATP-dependent 86Rb+ extrusion. This and other evidence suggested that active transport occurs via electroneutral H+in for K+out exchange. 86Rb+ exchange (uptake) in the proteoliposomes demonstrated both saturable and nonsaturable components. At a K0.5 = 1.5 mM, saturable 86Rb+ uptake accounted for about 90% of Rb+ influx. The vanadate-sensitive cation exchange indicated that the ATPase was reconstituted asymmetrically into the proteoliposomes (70% cis-/30% trans-vanadate site). 86Rb+ exchange was inhibited by ATP and stimulated about 2-fold by low Mg2+ and 5 mM phosphate. These ligand effects and the demonstration of comparable rates of passive exchange and active Rb+ efflux suggest that passive K+ exchange is not severely limited by a K+-occluded enzyme form in the H,K-ATPase. A model compatible with this hypothesis is suggested.

Adenosine Triphosphatases↗
PubMed1979

Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity.

Preparations of intestinal epithelial cell basal lateral plasma membranes were analyzed with free flow electrophoresis and density perturbation with digitonin. The initial basal lateral membrane preparations were obtained by equilibrium density gradient centrifugation after two different schemes of homogenization and differential sedimentation (A.K. Mircheff, C.H. van Os, and E.M. Wright. 1978. Membr. Biochem. 1:177, and A.K. Mircheff, S.D. Hanna, M.W. Walling, and E.M. Wright. 1979. Prep. Biochem. 9:33. In these preparations, Na,K-ATPase, a marker for the basal lateral mambrane, was purified 16- to 18-fold over the initial homogenate. The preparations were also enriched in NADPH-cytochrome c reductase, alkaline phosphatase, acid phosphatase, and galactosyltransferase. Both free-flow electrophoresis, which separates on the basis of surface charge, and density perturbation with digitonin, which depends on a specific interaction of digitonin with cholesterol-rich membranes, resolved the preparation into three populations of particles. The major population, which represented basal lateral membranes purified 20- to 32-fold with respect to the initial homogenate, contained Na,K-ATPase, alkaline phosphatase, adenylate cyclase, and acid phosphatase. A second population was defined by its content of NADPH-cytochrome c reductase, and the third was defined by its content of galactosyltransferase. Guanylate cyclase appeared to be partitioned between the Na,K-ATPase-rich and NADPH-cytochrome c reductase-rich populations. Galactosyltransferase is also present in fractions which contain the Na,K-ATPase-rich membranes, but the present data cannot exclude the possibility of spillover by the adjacent, galactosyltransferase-rich population. This work emphasizes the importance of multiple, physical criteria for purity in the isolation of subcellular components.

Animals↗
PubMed1979

Induction of a chloride conductance in gastric vesicles by limited trypsin or chymotrypsin digestion or ageing.

Transport activity of the hog gastric (H+ + K+)-ATPase system was measured either as the formation of proton gradient using the dye probe acridine orange or as the formation of a proton diffusion potential using the cyanine dye 3,3'-diethyloxdicarbocyanine iodide in the presence of the protonophore tetrachlorosalicylanilide. The development of these gradients has been compared in K+ media in the presence of either Cl- or SO4-2 as the anionic species. This comparison of proton diffusion potential formation to proton gradient formation has been used to demonstrate that a Cl- conductance in this vesicular system results from limited enzymic digestion with either trypsin or alpha-chymotrypsin from the ageing process itself. The possible significance of this finding is discussed.

Acridine Orange↗
PubMed1979

Transport characteristics of frog gastric membranes.

ATP-induced transport by fractions of frog gastric microsomes prepared either by density gradient centrifugation of by free flow electrophoresis were K+ dependent and hence considered due to a K+-activated ATPase. Significant activity of this enzyme was, however, only found in the anodic peak of the free flow electrophoretic separation, which in addition to separating transporting from non-transporting particles, also separated membranes containing a phosphorylatable peptide (Mr=105 000) region as the major peptide on SDS-polyacrylamide gel electrophoresis from those containing a peptide (Mr=44 000) on SDS-polyacrylamide gel electrophoresis. H+ uptake, measured either by acridine orange or 3,3'-diethyloxadicarbocyanine + tetrachlorosalicylanilide absorbance changes was dependent on K+ intravesicularly. Using 86Rb+, active extrusion of the cation followed ATP addition. SCN-, an inhibitor of acid secretion did not affect the latter, but blocked signals due to H+ uptake, in contrast to mammalian preparations.

Adenosine Triphosphatases↗
PubMed1978

Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles.

The ATP-dependent uptake of H+ by hog gastric parietal cell vesicles was quantitated by using the pH indicator dyes bromcresol green and malachite green, the weak bases, aminopyrine and 9-aminoacridine, and the pH electrode. A K+-dependent H+ uptake was found, with a significant difference between the quantity of H+ disappearing from the medium (deltaHo) and the quantity appearing inside the vesicle (deltaHi). 9-Aminoacridine gave a lower value for the deltaHi than any of the other probes. Probes of potential such as diethyloxadicarbocyanine or oxonol dyes showed that only secondary diffusion potentials occurred during H+ uptake and that the cationic dyes in the presence of protonophores could also be used to quantitate H+ uptake. The potential in the presence of protonophore indicated a deltaHi greater than that found with the other probes. Binding sites for acridine orange were generated either by ATP or an artificial pH gradient and corresponded to the deltaHi indicated by aminopyrine. SCN- (30mM) only partially inhibited the H+ gradient, and this, coupled with the failure to detect the physiological deltapH of 6.6, indicated that these vesicles may be an incomplete model of gastric acid secretion.

Adenosine Triphosphate↗
PubMed1977

Proton transport by gastric membrane vesicles.

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H+ uptake into an osmotically sensitive space. The apparent Km for ATP was 1.7-10(-4) M in the absence of a K+ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ and inhibited by ATPase inhibitors such as N,N'-dicylclohexyl-carbodiimide. Maximal H+ uptake occurs with an outward K+ gradient but the minimal apparent KA is found in the absence of a K+ gradient. The pH optimum for H+ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphroylation of the enzyme, but is considerably less than the pH maximum of the K+ dependent dephosphorylation. In the presence of an inward K+ gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H+ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K+ conductance but a measurable H+ conductance, hence a K+ gradient can produce an H+ gradient in the presence of valinomycin. The uptake and spontaneous leak of H+ are temperature sensitive with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H+ uptake by these vesicles is probably due to a dimeric (H+ + K+)-ATPase and is probably non-electrogenic.

Adenosine Triphosphatases↗
PubMed1977

Metabolic and membrane aspects of gastric H+ transport.

Metabolic properties of dog gastric mucosa, investigated by substrate level measurements, implicate the Krebs cycle as the major energy-yielding metabolic pathway but are equivocal in terms of an ATP-based H+ secretion. Purification of gastric membranes by centrifugation and free flow electrophoresis results in a class of membrane vesicles enriched in K+-ATPase and capable of ATP-energized H+ uptake. Immunohistochemistry shows these to be derived from the parietal cell. H+ uptake by the vesicles is accompanied by K+ efflux, and movement of either ion is not potential-coupled. The simplest interpretation of these transport studies is uptake of KCl by the vesicles by passive diffusion followed by active H+:K+ exchange. In some respects, however, this model fails to conform to the expectations from in vitro studies. It may be, therefore, that another pump (i.e., redox) or another membrane component (i.e., Cl- conductance) is lost during purification. The properties of the vesicles are such, however, as to establish their role in H+ secretion by the stomach.

Adenosine Diphosphate↗
PubMed1976

A nonelectrogenic H+ pump in plasma membranes of hog stomach.

Differential and density gradient centrifugation were used to prepare a vesicular membrane fraction from hog gastric mucosa enriched 17-fold with respect to cation-activated ATPase and 5'-AMPase. Fractionation of the gradient material by free flow electrophoresis resulted in a fraction 35-fold enriched in cation-activated ATPase and essentially free of 5'-AMPase and Mg2+ATPase. The addition of ATP to either fraction resulted in H+ uptake and Rb+ efflux. The ionophoric and osmotic sensitivity showed that these ion movements were due to transport rather than binding. The cation selectivity sequences, substrate specificities and action of inhibitors indicated that the transport was a function of K+ATPase activity. The characteristics of the ATP-dependent enhancement of SCN- uptake and 8-anilinonapthalene-1-sulfonate fluorescence in the presence of valinomycin and the action of ionophores and lipid-permeable ions suggested that the energy dependent K+:H+ exchange was effectively nonelectrogenic. Thus these vesicles contain a nonelectrogenic (H+ + K+)-ATPase, hence acid secretion by the stomach is probably due to an ATP-dependent H+ + K+ exchange.

Adenosine Triphosphatases↗