Sequence motif in control regions of the H+/K+ ATPase alpha and beta subunit genes recognized by gastric specific nuclear protein(s).

A nuclear protein(s) from rat or pig stomach recognized a conserved sequence in the 5'-upstream regions of the rat and human H+/K(+)-ATPase alpha subunit genes. A gel retardation assay suggested that part of the binding site was located in the TAATCAGCTG sequence. No nuclear proteins capable of the binding could be detected in other tissues of rat (liver, brain, kidney, spleen and lung) or pig liver. The sequence motif (GATAGC) located 5'-upstream of the beta-subunit gene also seemed to be recognized by the same protein, because the binding of nuclear protein to the sequence motifs in the alpha and beta subunits was mutually competitive. Considering the sense-strand sequence of the binding motif in the alpha-subunit gene, we conclude that (G/C)PuPu(G/C)NGAT(A/T)PuPy is a core sequence motif for the gastric specific DNA binding protein (PCSF, parietal cell specific factor).     

Effect of calcium on the H+/K+ ATPase of hog gastric microsomes

Summary The K⁺-stimulated, ouabain-insensitive ATPase activity present in vesicles of microsomal fractions from hog gastric mucosa can be demonstrated in fresh preparations by adding Ca²⁺ (M range) to the incubation medium. Ca²⁺ effect is similar but not additive to the effect of gramicidin or freezing. High Ca²⁺ concentrations (1 mM) produce an inhibotory effect on the K⁺-stimulated ATPase activity. This effect, is not seen in the presence of gramicidin. Calcium increases the magnitude of ATP-driven H⁺ uptake in vesicles exposed to K⁺ for periods of time up to 60 min. At longer times of exposure (120 min) the response does not differ from controls. It is concluded that Ca²⁺ at low concentrations (m range) enhances the K⁺ permeability of the vesicular membrane. At higher concentrations (mm range), Ca²⁺ becomes inhibitory to the K⁺ permeability. A role for Ca²⁺ as a second messenger in stimulus-secretion coupling in the parietal cell is discussed.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

An electrogenic NA+/K+ pump in the choroid plexus.

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na+/K+ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 . 10(-5) cm-1 . s-1 expressed per cm2 of flat epithelium.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

The H+/ATP stoichiometry of the (H+ +K+)-ATPase of dog gastric microsomes

Gastric microsomal vesicles isolated from dog fundic mucosa were shown to be relatively ion tight and have a low level of proton permeability. The H+ translocase, basal ATPase and K+-activated ATPase activities of these vesicles were measured and the H+/ATP stoichiometry calculated using either the total K+-ATPase or the K+-stimulatable component (total K+-ATPase--basal ATPase). The former estimations consistently gave stoichiometric of approximately one, whereas the use of only the K+-stimulatable component gave widely differing values. Measurement of the dephosphorylation of the enzyme under basal conditions revealed both a labile and a stable phosphoenzyme component. The rate of decay of the labile component completely accounted for the basal ATPase activity observed. We conclude that the basal ATPase associated with our preparations is a spontaneous dephosphorylation of the phosphoenzyme occurring in the absence of K+ and that the H+/ATP stoichiometry of the gastric ATPase is one.     

The H+/ATP stoichiometry of the (H+ + K+)-ATPase of dog gastric microsomes

Gastric microsomal vesicles isolated from dog fundic mucosa were shown to be relatively ion tight and have a low level of proton permeability. The H⁺ translocase, basal ATPase and K⁺-activated ATPase activities of these vesicles were measured and the H⁺/ATP stoichiometry calculated using either the total K⁺-ATPase or the K⁺-stimulatable component (total K⁺-ATPase—basal ATPase). The former estimations consistently gave stoichiometric of approximately one, whereas the use of only the K⁺-stimulatable component gave widely differing values. Measurement of the dephosphorylation of the enzyme under basal conditions revealed both a labile and a stable phosphoenzyme component. The rate of decay of the labile component completely accounted for the basal ATPase activity observed. We conclude that the basal ATPase associated with our preparations is a spontaneous dephosphorylation of the phosphoenzyme occurring in the absence of K⁺ and that the H⁺/ATP stoichiometry of the gastric ATPase is one.     

Anti-ulcerogenic and proton pump (H+, K+ ATPase) inhibitory activity of Kolaviron from Garcinia kola Heckel in rodents

Anti-ulcer potential and proton pump inhibitory activity of kolaviron (KV) isolated from Garcinia kola Heckel has been evaluated using different ulcer models. Cold-restraint (CRU), aspirin (ASP), alcohol (AL), pyloric ligation (PL) induced gastric ulcer models were used to assess anti-ulcerogenic activity of KV in rats. Effects of KV on gastric juice for free and total acidity, peptic activity and mucin secretion were also evaluated. The H+, K+-ATPase activity was assayed in gastric microsomes, spectrophotometrically. Results of this study showed that KV (200 mg/kg) reduced the incidence of ulcers in CRU (69.0%), PL (67.6%), ASP (68.6%) and AL (51.5%). Reductions were also observed in free acidity (32.6%), total acidity (56.2%) and peptic activity (35.4%) with increase in mucin secretion by 40.1%. KV inhibited the H+,K+-ATPase activity with IC50 of 43.8 microg/ml compared with omeprazole with IC50 of 32.3 microg/ml. KV showed both cytoprotective and anti-secretory potentials against peptic ulcer models, and a proton pump inhibitory activity. KV may emerge as a potent anti-ulcer compound.     

H+/ATP stoichiometry for the gastric (K++H+)-ATPase

Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H⁺/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H⁺/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K⁺ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H⁺/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K⁺+H⁺)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.     

K+-stimulated p-nitrophenyl phosphatase is not a partial reaction of the gastric (H+ + K+)-transporting ATPase. Evidence supporting a new model for the univalent-cation-transporting ATPase systems.

Studies with intact and lysed gastric microsomal vesicles demonstrate that there are two pNPP (p-nitrophenyl phosphate)-and one ATP-hydrolytic sites within the gastric H+, K+-ATPase [(H+ + K+)-transporting ATPase] complex. Whereas the ATPase site is located exclusively on the vesicle exterior, the pNPPase sites are distributed equally on both sides of the bilayer. Competition by ATP for the pNPPase reaction on the vesicle exterior suggests that both ATP and pNPP are hydrolysed at the same catalytic site present at the outside surface of the intact vesicles. However, a biphasic inhibition of the K+-pNPPase (K+-stimulated pNPPase) by ATP in the lysed vesicles suggest the pNPPase site of the vesicle interior to have very low affinity (Ki approximately equal to 1.2 mM) for ATP compared with the vesicle exterior (Ki approximately equal to 0.2 mM). Studies with spermine, which competes with K+ for the K+-pNPPase reaction without inhibiting the H+, K+-ATPase, suggest there are two separate K+ sites for the pNPPase reaction and another distinct K+ site for the ATPase reaction. In contrast with the K+ site for the ATPase, which is located opposite to the catalytic site across the bilayer, both the K+ and the catalytic site for the pNPPase are located on the same side. The data clearly demonstrate that the pNPPase is not a manifestation of the phosphatase step of the total H+, K+-ATPase reaction. The K+-pNPPase associated with the Na+, K+-ATPase also has properties strikingly similar to the gastric K+-pNPPase system, suggesting a resemblance in the basic operating principle of the two ion-transporting enzymes. A unified model has been proposed to explain the present data and many other observations reported in the literature for the ATPase-mediated transport of univalent cations.     

Monoclonal antibodies for the structural analysis of the Na+/H+ antiporter NhaA from Escherichia coli

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand. Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins. The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.     

The H+/ATP transport ratio of the (K+ + H+)-ATPase of pig gastric membrane vesicles

Various values have been reported for the H+/ATP transport ratio of the (K+ + H+)-ATPase of the gastric parietal cell: 4, 2 and 1. We have, therefore, reinvestigated this matter with a vesicle preparation isolated from pig gastric mucosa. The vesicles are suspended in glycylglycine buffer (pH 6.11) at 22 degrees C, and incubated until equalization of the K+ concentration inside and outside (75 mM). After addition of ATP, the initial rates of H+ uptake and ATP hydrolysis are then measured. Proton uptake is inhibited in the absence of K+ or in the presence of nigericin. The K0.5 value for proton transport is 154 microM and the Km value for ATP hydrolysis is 61 microM. The Lineweaver-Burk plot for ATP hydrolysis vs. ATP concentration is linear with a Vmax of 5.5 nmol/mg protein per s, but that for H+ uptake is not. Thus with increasing ATP concentration (6.7 to 1670 microM) the transport ratio increases from 0.3 to 1.8. Extrapolation to infinite ATP concentration gives a value of 1.89. (S.E. 0.13, N = 5) and a Hill coefficient of n = 1.21 (S.E. 0.06, N = 5) implying that the true transport ratio is 2 H+/ATP with positive cooperativity between the protons.     

Comparison of five antisecretory agents acting via gastric H+/K+-ATPase.

Lansoprazole(L), pantoprazole (P), rabeprazole and RO-18-5364 (RO) are new benzimidazole derivatives which rival omeprazole (O) as proton pump inhibitors (PPIs) for treatment of ulcer disease. In this study, we compared the effects of these compounds on acid secretion and determined their relative potencies in relation to their effect on [14C]-aminopyrine (AP) accumulation in isolated gastric glands. Inhibition of AP (1.2 microCi x mL(-1)) accumulation was measured in rabbit isolated gastric glands. dbcAMP (1 mmol; stimulant of acid secretion) and Ro 20-1724 (0.1 mmol; a phosphodiasterase inhibitor) were added to the Eppendorf tubes containing the PPIs and AP and dose-response curves were done for each drug after incubating for 5, 10 and 20 min at 37 degrees C and AP accumulation was determined using a scintillation counter. All the PPIs significantly (P < 0.001) inhibited acid secretion as demonstrated by the inhibition of AP accumulation in the isolated gastric glands. Minimum inhibition occurred at a concentration of 0.001 micromol for lansoprazole and omeprazole, 0.01 micromol for rabeprazole and RO 18-5364 and 0.02 micromol for pantoprazole. No differences were observed between PPIs with regards to the maximum inhibition they produce. When expressed as a percentage inhibition of control at 10-min incubation and at concentrations of 1 micromol, L showed 85.6 +/- 0.5, O 87 +/- 0.5, P 83.2 +/- 1.1, R 86.4 +/- 1.1 and RO 87.8 +/- 1.9 inhibition respectively. When comparing the IC50 values, their relative potencies were different. Maximum potency was shown by L (0.007 micromol) > O (0.012 micromol) > R (0.018 micromol) > RO (0.034 micromol) > P (0.050 micromol). All the new PPIs showed different potencies as inhibitors of acid secretion as evident from their IC50s. Extensive ulcer healing trials demonstrated comparable efficacy with a number of studies indicating that symptoms relief are more rapid with P and L, while in this study L appeared to be the most potent in inhibiting AP accumulation in the isolated gastric glands.     

Inhibition of H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase by strophanthidin.

Studies of the effect of strophanthidin on H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase activities are reported. Inhibition observations and kinetic results suggest the existence of a common digitalis aglycone binding site located on the extracellular surface of the enzyme, which is affected competitively by the binding of potassium to H(+)-transporting ATPase, Ca(2+)-transporting ATPase, as well as H+/K(+)-transporting ATPase and Na+/K(+)-transporting ATPase. This may lead to a better understanding of the mechanism of the pharmacological action of cardiac glycosides and imply the possibility that the positive inotropic effect may result from the inhibition of both Ca(2+)-transporting ATPase and Na+/K(+)-transporting ATPase.     

Role of membrane-associated thiol groups in the functional regulation of gastric microsomal (H+ + K+)-transporting ATPase system.

The distribution of free thiol groups associated with the membrane proteins of the purified pig gastric microsomal vesicles was quantified, and the relation of thiol groups to the function of the gastric (H+ + K+)-transporting ATPase system was investigated. Two different thiol-specific agents, carboxypyridine disulphide (CPDS) and N-(1-naphthyl)maleimide (NNM) were used for the study. The structure-function relationship of the membrane thiol groups was studied after modification by the probes under various conditions, relating the inhibition of the (H+ + K+)-transporting ATPase to the ATP-dependent H+ accumulation by the gastric microsomal vesicles. On the basis of the extent of stimulation of the microsomal (H+ + K+)-transporting ATPase in the presence and absence of valinomycin (val) about 85% of the vesicles were found to be intact. CPDS at 1 mM completely inhibits the valinomycin-stimulated ATPase and the associated p-nitrophenyl phosphatase with a concomitant inhibition of vesicular H+ uptake. Both the enzyme and dye-uptake activities were fully protected against CPDS inhibition when the treatment with CPDS was carried out in the presence of ATP. ATP also offered protection (about 65%) against NNM inhibition of the (H+ + K+)-transporting ATPase system and vesicular H+ uptake. Under similar conditions ATP also protected about 10 and 6 nmol of thiol groups/mg of protein respectively from CPDS and NNM reaction. Our data suggest that the thiol groups on the outer surface of the vesicles are primarily involved in gastric (H+ + K+)-transporting ATPase function. Furthermore, at least about 15% of the total microsomal thiol groups appear to be associated with the ATPase system. The data have been discussed in terms of the structure-function relationship of gastric microsomes.     

Reconstitution of the mitochondrial non-selective Na+/H+ (K+/H+) antiporter into proteoliposomes

Mitochondria contain two Na+/H+ antiporters, one of which transports K+ as well as Na+. The physiological role of this non-selective Na+/H+ (K+/H+) antiporter is to provide mitochondrial volume homeostasis. The properties of this carrier have been well documented in intact mitochondria, and it has been identified as an 82,000-dalton inner membrane protein. The present studies were designed to solubilize and reconstitute this antiporter in order to permit its isolation and molecular characterization. Proteins from mitoplasts made from rat liver mitochondria were extracted with Triton X-100 in the presence of cardiolipin and reconstituted into phospholipid vesicles. The reconstituted proteoliposomes exhibited electroneutral 86Rb+ transport which was reversibly inhibited by Mg2+ and quinine with K0.5 values of approximately 150 and 300 microM, respectively. Incubation of reconstituted vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of 86Rb+ uptake into proteoliposomes. Incubation of vesicles with [14C]dicyclohexylcarbodiimide resulted in labeling of an 82,000-dalton protein. These properties, which are also characteristic of the native Na+/H+ (K+/H+) antiporter, lead us to conclude that this mitochondrial carrier has been reconstituted into proteoliposomes with its known native properties intact.     

Ca2+/calmodulin-dependent protein kinase II isoenzymes gamma and delta are both present in H+/K+-ATPase-containing rabbit gastric tubulovesicles.

Ca2+/calmodulin-dependent protein kinase II is thought to participate in M3 muscarinic receptor-mediated acid secretion in gastric parietal cells. During acid secretion tubulovesicles carrying H+/K+-ATPase fuse with the apical membrane. We localized Ca2+/calmodulin-dependent protein kinase II from highly purified rabbit gastric tubulovesicles using Ca2+/calmodulin-dependent protein kinase II isoform-specific antibodies, in vitro phosphorylation and pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II activity by the potent Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. The presence of Ca2+/calmodulin-dependent protein kinase II in tubulovesicles was shown by immunoblot detection of both Ca2+/calmodulin-dependent protein kinase II-gamma (54 kDa) and Ca2+/calmodulin-dependent protein kinase II-delta (56.5 kDa). The immunoprecipitated Ca2+/calmodulin-dependent protein kinase II from tubulovesicles showed Ca2+/calmodulin-dependent protein kinase activity by phosphorylating autocamtide-II, a specific synthetic Ca2+/calmodulin-dependent protein kinase II substrate. KN-62 inhibited the in vitro autophosphorylation of tubulovesicle-associated Ca2+/calmodulin-dependent protein kinase II (IC50 = 11 nM). During the search for potential Ca2+/calmodulin-dependent protein kinase II substrates we identified different proteins associated with tubulovesicles, such as synaptophysin and beta-tubulin immunoreactivity, which were identified using specific antibodies. These targets are known to participate in intracellular membrane traffic. Ca2+/calmodulin-dependent protein kinase II is thought to play an important role in regulating tubulovesicular motor activity and therefore in acid secretion.     

The identification of a proton pump on vacuoles of the yeast Saccharomyces carlsbergensis. ATPase is electrogenic H+-translocase

A cadmium-binding protein rich in cysteine and acidic amino acid residues was isolated from roots of Agrostis gigantea. The molar ratio of cysteine to cadmium was 2.7:1. Electronic absorption and circular dichroism measurements were characteristic of cadmium-thiolate coordination. The cadmium-binding centre in the plant protein was strikingly similar to that of the well characterised vertebrate cadmiumthioneins.