Search PubMed⌕ Search

Biomedical subjects

E Rabon

Publications and source records attributed to E Rabon.

At least 19 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↗
PubMed1983

Vanadate binding to the gastric H,K-ATPase and inhibition of the enzyme's catalytic and transport activities.

Vanadate inhibition of the catalytic and transport activities of the gastric magnesium-dependent, hydrogen ion transporting, and potassium-stimulated adenosinetriphosphatase (EC 3.6.1.3) (H,K-ATPase) has been studied. The principal experiment observations are the following: (1) Inhibition of adenosine 5'-triphosphate (ATP) hydrolysis is biphasic. Vanadate binding with a stoichiometry of 1.5 nmol mg-1 approximately halves K+-stimulated ATPase activity at physiological temperature. The remaining activity is inhibited by binding an additional 1.5 nmol mg-1 vanadate with lower apparent ions bind specifically to gastric vesicles with two affinities. Vanadate binding in the presence of nucleotide is compatible with competition for the kinetically defined high-affinity and low-affinity ATP sites. (3) Vanadate inhibits phosphoenzyme formation and the K+-stimulated p-nitrophenyl phosphatase activity of the enzyme monophasically. A maximum of 1.5 nmol mg-1 acid-stable phosphoenzyme is formed. The half-time for vanadate dissociation from the site that inhibits p-nitrophenyl phosphate hydrolysis is 5 min (4) At most, 3 nmol mg-1 vanadate is required to inhibit proton transport. The simplest interpretation of the data is that vanadate inhibits the H,K-ATPase by binding competitively with ATP at two catalytic sites. Different catalytic mechanisms at the high-affinity and low-affinity sites are suggested by the different stoichiometries found for vanadate binding and phosphoenzyme formation.

Adenosine Triphosphatases↗
PubMed1983

Proton secretion by the gastric parietal cell.

The parietal cell occupies a unique niche among eukaryotic cells in that it develops a proton gradient of more than 4 million-fold across the membrane of the secretory canaliculus. At rest, the cell is still able to develop a proton gradient across intracellular membranes, such that the acid compartment has a pH of less than 4. Acidification depends on the simultaneous presence of ATP, K+ and Cl- as demonstrated in permeabilized cells. With acidification of the luminal side of the proton pump, there is a corresponding alkalinization of the cytosolic face as revealed by carboxyfluorescein fluorescence enhancement. Disposal of the resultant alkali depends on carbonic anhydrase activity and the functioning of a coupled Na+:H+ and Cl-:OH-antiport across the basal lateral membrane. Accordingly, with secretion there is an increased cellular Cl- level, which is exported across the apical membrane in association with K+. The Na+ pump dependent secretion of KCl across this membrane is one of the major sites of the gastric ATPase. Membranes isolated from secreting tissue contain a KCl permeation pathway largely absent from membranes isolated from resting tissue. The pump itself acts as an H+ for K+ exchange ATPase which is most probably composed of at least two peptides of 100 000 Mr. That catalytic cycle consists of formation and breakdown of a covalent aspartyl phosphate. Formation of the intermediate depends on loss of K+ from cytosolic binding sites, and breakdown of the intermediate depends on K+ binding to the luminal face of the enzyme. During breakdown, an acid labile E . P is formed, and, at high ATP concentrations, loss of this form of the enzyme is probably the rate limiting step.

Adenosine Triphosphatases↗
PubMed1982

ATP/ADP exchange activity of gastric (H+ +K+)-ATPase.

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+.

Adenosine Diphosphate↗
PubMed1982

Ion pathways in renal brush border membranes.

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na+/H+ or Li+/H+ exchange was demonstrated by diluting Na2SO4 or Li2SO4 loaded vesicles into Na+-or Li+-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K+ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na+ conductance smaller than electroneutral Na+/H+ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na+ conductance greater than the H+ conductance was suggested. K+ gradients also induced changes of pH, at about 10% of the Na+ or Li+ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K+ was a low affinity substrate for the Na+/H+ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K+ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles has a low K+ conductance. Inward Cl- gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl- conductance greater than the H+ conductance and a Cl-/OH- exchange were present. The rate of Na+/H+ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent Km for Na+ or Li+ of about 10 mM and an EA of 14.3 kcal per mol. A 61-fold Na2SO4 gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower delta pH even in the presence of gramicidin showing that the H+ and Cl- pathways could alter the effect of the Na+/H+ exchange.

Acridine Orange↗
PubMed1982

Proton/hydroxyl transport in gastric and intestinal epithelia.

Proton transport across the plasma membrane of the gastrointestinal epithelium occurs by various pathways. There is the permeability of H+ across the lipid components of the membranes, probably of minor significance at physiological pH, but at the pH of the secretory surface of the parietal cell a factor that cannot be neglected. Transport of H+ dependent on the protein components of the plasma membrane involves various mechanisms. For example Na+ :H+ or Cl- :HCO-3 antiport (exchange) are generally electroneutral mechanisms (i.e., neither affected by potential gradients nor affecting membrane conductance) that are widely distributed throughout the body. Plasma membranes may contain proton or bicarbonate conductances (i.e., gradients of either ion may be determined by the potential across the membrane). This type of pathway is often of minor significance, hence the electrical component of hydrogen ion gradients across the plasma membrane can often be neglected. In the case of the gastric parietal cell, proton transport depends on the activity of a specific ATPase. This ATPase may be present elsewhere in the intestinal tract. This review will consider many of these proton pathways. In the case of brush border pathways, some of the data presented on Na+ :H+ antiport wil be derived from studies done on renal brush border rather than those of the small intestine, on the assumption that the properties of the antiporter are similar in the two tissues.

Adenosine Triphosphatases↗