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PubMed1999

Molecular cloning and chromosomal assignment of the porcine 54 and 56 kDa vacuolar H(+)-ATPase subunit gene (V-ATPase).

Vacuolar proton-translocating ATPases (V-ATPase) are multisubunit enzyme complexes located in the membranes of eukaryotic cells regulating cytoplasmic pH. So far, nothing is known about the genomic organization and chromosomal location of the various subunit genes in higher eukaryotes. Here we describe the isolation and analysis of a cDNA coding for the 54- and 56-kDa porcine V-ATPase subunit alpha and beta isoforms. We have determined the genomic structure of the V-ATPase subunit gene spanning at least 62 kb on Chromosome (Chr) 4q14-q16. It consists of 14 exons with sizes ranging from 54 bp to 346 bp, with a non-coding first exon and an alternatively spliced seventh exon leading to two isoforms. The 5' end of the V-ATPase cDNA was isolated by RACE-PCR. The V-ATPase alpha isoform mRNA, lacking the seventh exon, has an open reading frame of 1395 nucleotides encoding a hydrophilic protein of 465 amino acids with a calculated molecular mass of 54.2 kDa and a pI of 7.8, whereas the beta isoform has a length of 1449 nucleotides encoding a protein of 483 amino acids with a calculated molecular mass of 55.8 kDa. Amino acid and DNA sequence comparison revealed that the porcine V-ATPase subunit exhibits a significant homology to the VMA13 subunit of Saccharomyces cerevisiae V-ATPase complex and V-ATPase subunit of Caenorhabditis elegans.

Amino Acid Sequence↗

PubMed2001

Bile canalicular contraction and dilatation in primary culture of rat hepatocytes--possible involvement of two different types of plasma membrane Ca(2+)-Mg(2+)-ATPase and Ca(2+)-pump-ATPase.

Increasing evidence has indicated that bile canalicular contraction is mediated by the nonmuscular Ca(2+)-calmodulin-actomyosin system, and the contraction facilitates canalicular bile flow. The aim of the present study was to examine, by electron cytochemistry, how the expression of two types of plasma membrane Ca(2+)-ATPase, i.e., Ca(2+)-Mg(2+)-ATPase and Ca(2+)-pump-ATPase, is related to the dynamic changes of bile canalicular contraction. Hepatocytes isolated from male Wistar rat liver by collagenase perfusion were cultured to form a primary monolayer. The canalicular dynamics in the couplets and triplets were analyzed by time-lapse cinematography. The Ca(2+)-Mg(2+)-ATPase activity was identified by the electron cytochemical method of Ando. Ultrastructural localization of Ca(2+)-pump-ATPase was examined by immunogold electron microscopy. We found that cytochemical reaction products showing the presence of Ca(2+)-Mg(2+)-ATPase activity were localized on the luminal side of the bile canalicular membranes. Immunogold particles, indicating the presence of Ca(2+)-pump-ATPase, were located mainly on the cytoplasmic side of the bile canalicular membranes. The expression of both Ca(2+)-ATPases on the canalicular membranes was enhanced during the contracting stage of bile canaliculi, whereas their expression was diminished in the dilating stage. We conclude that two different types of bile canalicular Ca(2+)-ATPase may be involved in the regulation of canalicular contractility to control the extrusion of intracytoplasmic free calcium ions into the canalicular lumen.

Animals↗

PubMed1994

Evidence that both Ca(2+)-ATPase and (Ca2+ + Mg2+)-ATPase activities in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme molecule.

1. Evidence was obtained that activities of both low-affinity Ca(2+)-ATPase and high-affinity (Ca2+ + Mg2+)-ATPase in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme. 2. Two solubilized ATPases were purified by four steps of HPLC; and both activities eluted at the same fractions from each column, and the specific activity ratio of the two enzymes at each step was constant. 3. By non-denaturing PAGE, the final preparation gave a single band for both protein staining and activity staining for the two ATPases; and the Ca(2+)-ATPase activity comigrated with that of (Ca2+ + Mg2+)-ATPase. 4. In SDS-PAGE, each activity staining for the ATPases also gave a single band, and both activities comigrated. 5. These findings suggest that Ca(2+)-ATPase and (Ca2+ + Mg2+)-ATPase are a single enzyme.

Animals↗

PubMed2004

Protective effect of L-cysteine and glutathione on the modulated suckling rat brain Na+, K+, -ATPase and Mg2+ -ATPase activities induced by the in vitro galactosaemia.

UNLABELLED: Galactosaemia is an inborn error of galactose (Gal) metabolism characterized by irreversible brain damage. The aim of this study was to evaluate whether the antioxidants L-cysteine (Cys) and the reduced glutathione (GSH) could reverse the alterations of brain total antioxidant status (TAS) and the modulated activities of the enzymes Na+,K+ -ATPase and Mg2+ -ATPase in in vitro galactosaemia. Mixture A (mix. A: galactose-1-phosphate (Gal-1-P, 2mM) plus galactitol (Galtol, 2mM) plus Gal (4mM) = classical galactosaemia) or Mixture B (mix. B: Galtol (2mM) plus Gal (1mM) = galactokinase deficiency galactosaemia) were preincubated in the presence or absence of Cys (0.83mM) or GSH (0.83 mM) with whole brain homogenates of suckling rats at 37 degrees C for 1h. TAS and the enzyme activities were determined spectrophotometrically. The preincubation of brain homogenates with mix. A or mix. B resulted in a decrease of TAS to 30% (P < 0.01), while the presence of Cys or GSH increased TAS to 20% (P < 0.01) and 60% ( P < 0.001), respectively. The antioxidants reversed the inhibited Na+,K+ -ATPase by mix. A or mix. B and the stimulated Mg2+ -ATPase by mix. B to control values, whereas no effect was observed on the enormously activated Mg2+ -ATPase by mix. A. CONCLUSIONS: (a) Gal and its derivatives may produce free radicals in the suckling rat brain, reported for first time, (b) Na+,K+ -ATPase inhibition and Mg2+ -ATPase activation are probably due to the oxidative stress from the above compounds, (c) Cys or GSH could play a protective role reversing the inhibited Na+,K+ -ATPase toward normal in in vitro galactosaemia and (d) the addition of the above antioxidants may reduce the consequences of brain Mg2+ -ATPase activation by Gal and Galtol in galactokinase deficiency galactosaemia.

Animals↗

PubMed2001

Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states.

Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases.

Adenosine Triphosphatases↗

PubMed2001

Na+,K+-ATPase and Ca2+-ATPase activities in the cochlear lateral wall following surgical induction of hydrops.

Na+,K+-ATPase and Ca2+-ATPase activities have not been studied quantitatively in the cochlea affected by endolymphatic hydrops. The present study was designed to measure quantitatively the Na+,K+-ATPase and Ca2+-ATPase activities in the cochlear lateral wall and the threshold of auditory brainstem response (ABR) for guinea pigs in the early stages (=2 months) of experimentally induced endolymphatic hydrops. A significant negative association was demonstrated between Ca2+-ATPase activity and the change in ABR threshold for hydropic cochleae (P=0.014), but not for control cochleae (P=0.123), although no such significant association was revealed between Na+,K+-ATPase activity and any change in ABR threshold for both hydropic cochleae (P=0.751) and control cochleae (P=0.352). A significant increase in Ca2+-ATPase activity in the cochlear lateral wall was observed for the hydropic ear, in which normal ABR thresholds were maintained, as compared to the control ear. On the contrary, a mild decrease in Ca2+-ATPase activity in the cochlear lateral wall was observed for the hydropic ear, in which ABR thresholds increased significantly. The present findings suggest that alterations of Ca2+-ATPase activity in the cochlear lateral wall may implicate disturbed calcium-homeostasis in the inner ear, resulting in hearing dysfunction in the early stages of experimentally induced endolymphatic hydrops.

Animals↗

PubMed1997

Mutations of pma-1, the gene encoding the plasma membrane H+-ATPase of Neurospora crassa, suppress inhibition of growth by concanamycin A, a specific inhibitor of vacuolar ATPases.

Concanamycin A (CCA), a specific inhibitor of vacuolar ATPases, inhibited growth of Neurospora crassa in medium adjusted to pH 7 or above. Mutant strains were selected for growth on medium containing 1.0 microM CCA. Sixty-four (of 66) mutations mapped in the region of the pma1 locus, which encodes the plasma membrane H+-ATPase. Analysis of V-ATPase activity in isolated vacuolar membranes from the mutant strains showed wild-type activity and sensitivity to CCA. In contrast, plasma membrane H+-ATPase activity in isolated plasma membranes from the mutants was reduced as compared with wild-type, and in four strains the activity showed increased resistance to vanadate. The most interesting change in the plasma membrane H+-ATPase was in kinetic behavior. The wild-type enzyme showed sigmoid dependence on MgATP concentration with a Hill number of 2.0, while the seven mutants tested exhibited hyperbolic kinetics with a Hill number of 1.0. One interpretation of these data was that the enzyme had changed from a functional dimer to a functional monomer. Mutation of the plasma membrane H+-ATPase did not confer resistance by preventing uptake of CCA. In the presence of CCA both wild-type and mutant strains were unable to accumulate arginine, failed to concentrate chloroquine in acidic vesicles, and exhibited gross alterations in hyphal morphology, indicating that the CCA had entered the cells and inactivated the V-ATPase. Instead, we hypothesize that the mutations conferred resistance because the altered plasma membrane H+-ATPase could more efficiently rid the cell of toxic levels of Ca2+ or protons or other ions accumulated in the cytoplasm following inactivation of the V-ATPase by CCA.

Anti-Bacterial Agents↗

PubMed1999

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase.

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.

Adenosine Triphosphate↗

PubMed2001

Vacuolar H+-ATPase, but not mitochondrial F1F0-ATPase, is required for aluminum resistance in Saccharomyces cerevisiae.

It was recently shown that vacuolar ATPase and mitochondrial F1F0-ATPase activities are induced by aluminum (Al) in an Al-resistant cultivar of wheat, suggesting that induction of these enzymes could be an adaptive trait involved in Al resistance. To test this hypothesis, we used the Saccharomyces cerevisiae model system. In yeast, unlike wheat, the activity, transcript and protein levels of mitochondrial F1F0-ATPase, but not vacuolar ATPase, are induced by Al, while plasma membrane P-ATPase activity is inhibited. However, yeast vacuolar ATPase mutant strains are hypersensitive to Al, while F1F0-ATPase mutant strains exhibit wild-type growth. These data suggest that vacuolar ATPase activity is involved in Al resistance, with ATP required for this activity supplied by mitochondrial F1F0-ATPase or fermentation.

Aluminum↗

PubMed2001

The effects of the pre-treatment of intravenous nimodipine on Na(+)-K+/Mg+2 ATPase, Ca+2/Mg+2 ATPase, lipid peroxidation and early ultrastructural findings following middle cerebral artery occlusion in the rat.

Excessive calcium influx has been implicated in the pathophysiology of ischemic cerebral damage. The effects of nimodipine, a calcium antagonist, on the Na(+)-K+/MG+2 ATPase activity, Ca+2/Mg+2 ATPase, lipid peroxidation, and early ultrastructural findings were examined at the acute stage of ischemia in the rat brain. Ischemia was produced by permanent unilateral occlusion of the middle cerebral artery. In Group I, the rats which had no ischemia and not received medication were used for determining Na(+)-K+/Mg+2 ATPase, Ca+2/Mg+2 ATPase, the extent of lipid peroxidation by measuring the malondialdehyde content and normal ultrastructural findings. In Group II, the rats which had only subtemporal craniectomy without occlusion and received saline solution were used for determining the effect of the surgical procedure on the biochemical indices and ultrastructural findings. In Group III, the rats received saline solution following the occlusion in the same amount of nimodipine and in the same duration as used in Group IV. In Group IV, nimodipine pre-treatment 15 min before occlusion (microgram kg-1 min-1 over a 10 min period) was applied i.v. Na(+)-K+/Mg+2 ATPase and Ca+2/Mg+2 ATPase activities decreased significantly and promptly as early as 10 min and remained at a lower level than the contralateral hemisphere in the same group and at the normal level in Group I. Nimodipine pre-treatment immediately attenuated the inactivation of Na(+)-K+/Mg+2 ATPase (p < 0.05) but there was no change on Ca+2/Mg+2 ATPase activity (p < 0.05). Malondialdehyde content increased significantly in Group III following ischemia as early as 30 min. Nimodipine pre-treatment decreased the malondialdehyde level in Group IV (p < 0.05). This study supports the possibility that nimodipine pre-treatment effects the membrane stabilizing properties via inhibiting the lipid peroxidation and subsequently restoring some membrane bound and lipid dependent enzymes' activity such as Na(+)-K+/Mg+2 ATPase and the ultrastructural findings.

Animals↗

PubMed1999

Increased intracellular calcium and decreased activities of leucocyte Na+,K+-ATPase and Ca2+-ATPase in asthma.

The present study was carried out to determine the intracellular free calcium concentration ([Ca(2+)](i)) and the activity of its regulatory enzymes (Na(+),K(+)-ATPase and Ca(2+)-ATPase) in leucocytes. Levels of plasma lysophosphatidylcholine (LPC) were also measured. Then the relationship between these parameters and the clinical severity of asthma and bronchial reactivity was studied. Patients with asthma were divided into three groups: acute asthma (subjects in acute exacerbation), uncontrolled asthma (subjects currently symptomatic) and stable asthma (subjects currently asymptomatic). A group of normal subjects was also studied. Spirometry, specific airway conductance and bronchial reactivity measurements were carried out. The following biochemical parameters were studied in venous blood: leucocyte [Ca(2+)](i), Na(+), K(+)-ATPase and Ca(2+)-ATPase activities, and plasma LPC. Leucocyte [Ca(2+)](i) was increased and the activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase were decreased in patients with asthma. Plasma levels of LPC were also increased. These changes were observed to be greatest among asthmatics in acute exacerbation of asthma, and lesser in magnitude in patients with less severe asthma. The activities of both ATPases were found to have a significant positive correlation, and [Ca(2+)](i) and the levels of plasma LPC a significant negative correlation, with predicted forced expiratory volume in 1 s (FEV(1)). No significant correlation was observed between the biochemical parameters and bronchial reactivity. It is concluded that intracellular calcium homoeostasis is abnormal in asthma; specifically, the activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase are decreased. These abnormalities may modulate the clinical severity of asthma.

Acute Disease↗

PubMed1986

The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase.

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.

Bacillus↗

PubMed1993

Factors affecting the species-homologous and species-heterologous binding of mitochondrial ATPase inhibitor, IF1, to the mitochondrial ATPase of slow and fast heart-rate hearts.

We examined the effects of a variety of conditions upon the IF1-mediated inhibition of the ATPase in both intact and sonicated mitochondria and in IF1-depleted submitochondrial particles (SMP) in species-homologous and species-heterologous combinations of IF1 and ATPase. IF1-mediated ATPase inhibition occurred in intact rabbit heart mitochondria at low matrix pH and low membrane potential, but not in intact pigeon and rat heart mitochondria under the same conditions. IF1-mediated ATPase inhibition was, however, demonstrable in both the rabbit and pigeon heart systems in sonicated mitochondria incubated at low ionic strength. The rat heart system failed to exhibit significant IF1-mediated ATPase inhibition in either intact or sonicated mitochondria due to the low amount of IF1 present. When rabbit heart IF1-containing extracts were incubated with IF1-depleted rabbit heart SMP over a range of KCl concentrations, increasing the [KCl] to 100 mM had little effect on IF1-mediated ATPase inhibition. When pigeon heart IF1-containing extracts were incubated with IF1-depleted pigeon heart SMP under the same conditions, increasing [KCl] to 100 mM nearly completely blocked IF1-mediated ATPase inhibition. While the species-endogenous level of rat heart IF1 (i.e., 1x IF1) inhibited IF1-depleted rat heart SMP virtually not at all at any [KCl] examined, the 8x rat heart IF1 was nearly as inhibitory as the 1x rabbit heart IF1 at varying ionic strengths. When rabbit, pigeon, or rat heart IF1 was bound to rabbit versus pigeon IF1-depleted SMP, the effect of varying ionic strength on IF1-mediated ATPase inhibition was related to the species source of the IF1, not to the species source of the enzyme; 1x bovine heart IF1 purified to homogeneity behaved much like 1x crude rabbit heart IF1 when binding to either the rabbit or the pigeon heart enzyme. This suggests that an IF1-ATPase complex stabilizing factor such as has been isolated from baker's yeast cells in neither lacking in the pigeon heart system nor required for the more ionic-strength-resistant binding of IF1 observed in slow heart-rate mammalian heart mitochondria.

Adenosine Triphosphatases↗

PubMed1989

Lysosomal H+-translocating ATPase has a similar subunit structure to chromaffin granule H+-ATPase complex.

Subunit structure of the lysosomal H+-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H+-ATPase from chromaffin granules and chemical modification with N,N'-dicyclohexyl[14C]carbodiimide. The lysosomal H+-ATPase was irreversibly inhibited when incubated at 0 degrees C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretic mobility as the corresponding subunits of chromaffin granule H+-ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologically identical to the corresponding subunits of chromaffin granule H+-ATPase. Dicyclohexylcarbodiimide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H+-ATPase is a multimeric enzyme, whose subunit structure is similar to the chromaffin granule H+-ATPase. The subunit structure of other vacuolar H+-ATPases, revealed by cold inactivation and immunological cross-reactivity, is also presented.

Adrenal Glands↗

PubMed1992

Rapid purification and reconstitution of a plant vacuolar ATPase using Triton X-114 fractionation: subunit composition and substrate kinetics of the H(+)-ATPase from the tonoplast of Kalanchoë daigremontiana.

A rapid procedure for the purification and reconstitution into proteoliposomes of the H(+)-translocating ATPase of plant vacuolar membranes is reported. It involves fractionation of the tonoplast with Triton X-114, resolubilization of the ATPase with octyl glucoside in the presence of a mixture of phosphatidylcholine, phosphatidylserine and cholesterol (27:53:20, by weight), and removal of the detergent by gel-filtration. Starting with partially purified vacuolar membranes, the procedure can be accomplished in about 2 hours. It has been applied to the H(+)-ATPase from the crassulacean plant Kalanchoë daigremontiana, from which it yields vesicles with a specific ATPase activity of about 3 mumol/min per mg protein. The purified enzyme contains polypeptides of apparent molecular mass 72, 57, 48, 42, 39, 33 and 16 kDa; these polypeptides also co-sediment on centrifugation of the solubilized ATPase through glycerol gradients. The 16-kDa subunit is labelled with [14C]dicyclohexylcarbodiimide. There is no evidence for a larger ATPase subunit in this preparation. The reconstituted ATPase proteoliposomes undergo ATP-dependent acidification, which can be measured by quenching of the fluorescence of 9-aminoacridine. The initial rate of fluorescence quenching is a measure of the rate of H+ translocation, and is directly proportional to the vesicle protein concentration, so the preparation is suitable for studying the kinetics of the tonoplast H(+)-ATPase. The dependence of the rate of fluorescence quenching on the concentration of MgATP is well fitted by the Michaelis equation, with a Km value about 30 microM. ATP can be replaced by dATP, ITP, GTP, UTP or CTP, and Mg2+ by Mn2+ or Ca2+; kinetic parameters for these substrates are reported. In contrast, hydrolysis of MgATP shows complex kinetics, suggestive either of negative cooperativity between nucleotide-binding sites, or of two non-interacting catalytic sites. Both the hydrolytic and the H(+)-translocating activities of the proteoliposomes are inhibited by nitrate, though not in parallel, the latter activity being the more sensitive. Both activities are inhibited in parallel by bafilomycin A1, which does not produce complete inhibition; the bafilomycin-insensitive component has complex ATPase kinetics similar to those of the uninhibited enzyme.

Adenosine Triphosphate↗

PubMed1990

The presence of H+,K(+)-ATPase in the crypt of rabbit distal colon demonstrated with monoclonal antibodies against gastric H+,K(+)-ATPase.

Indirect evidence indicates the presence of an active H+/K+ antiporter for the secretion of acid in the distal colon. It was examined whether the H+/K+ antiporter in the rabbit distal colon was hydrogen-potassium-stimulated adenosine triphosphatase (H+,K(+)-ATPase), which acts as a proton pump in the gastric mucosa. For this purpose, four monoclonal antibodies against hog gastric H+,K(+)-ATPase were raised. Three monoclonal antibodies dose-dependently inhibited the ouabain-insensitive gastric ATP-ase activity. Antibody HK4001 completely inhibited the ATPase activity. In indirect immunofluorescence studies, all four monoclonal antibodies stained H+,K(+)-ATPase in gastric mucosae of various animal species. Two monoclonal antibodies including antibody HK4001 cross-reacted with H+,K(+)-ATPase located in crypts of the transverse and descending colon and rectum of rabbits. Because the other two antibodies did not cross-react with the H+,K(+)-ATPase in the colon, this colonic enzyme is similar but not identical to gastric H+,K(+)-ATPase. On the other hand, HK4001 and SCH 28080 did not inhibit ouabain-sensitive K(+)-dependent ATPase activity in the guinea pig distal colon, and the antibodies did not stain the enzyme in the tissue. Therefore, ouabain-sensitive H+/K+ antiporter in the guinea pig is not similar to ouabain-insensitive rabbit colonic H+,K(+)-ATPase.

Adenosine Triphosphatases↗

PubMed2006

Expression of gastric ghrelin and H(+)-K(+)-ATPase mRNA in weanling piglets and effect of ghrelin on H(+)-K(+)-ATPase expression and activity in gastric mucosal cells in vitro.

This study was designed to investigate the effect of ghrelin on gastric acid secretion in weaning piglets both in vivo and in vitro. Thirty newborn piglets were selected from six litters and on 28, 35 (weaning), 38, 42 and 45d of age, respectively, one piglet from each litter was killed and the mucosal tissue from gastric fundus was collected for detecting ghrelin mRNA as well as H(+)-K(+)-ATPase mRNA expression and activity. Primary cultures of gastric mucosal cells from 5-week-old weaning piglets were challenged with 3x10(-5), 3x10(-4), 3x10(-3), 3x10(-2) and 3x10(-1)nmol/ml h-ghrelin, respectively, for 4h in order to further clarify the effect of ghrelin on gastric H(+)-K(+)-ATPase mRNA expression and activity. Ghrelin mRNA expression in gastric fundus kept stable from 28d to 42d, followed by a sudden increase on 45d, exhibiting a peak that was significantly higher than any other age groups investigated. H(+)-K(+)-ATPase activity and mRNA expression showed similar trends of increase with slightly different timing. H(+)-K(+)-ATPase mRNA expression tended to increase on 42d, while H(+)-K(+)-ATPase activity started to rise from 35d, but neither of them reached significantly higher levels until 45d. In vitro, ghrelin significantly increased H(+)-K(+)-ATPase activity of gastric mucosal cells at 3x10(-4), 3x10(-3), and 3x10(-2)nmol/ml, but augmented H(+)-K(+)-ATPase mRNA expression only at 3x10(-4)nmol/ml. The results indicate that ghrelin mRNA expression is up-regulated 10 days after weaning in the gastric fundus of piglets, coinciding with the increase of H(+)-K(+)-ATPase mRNA expression and activity. Ghrelin acts on gastric mucosal cells to stimulate both mRNA expression and activity of H(+)-K(+)-ATPase in vitro.

Animals↗

PubMed2001

Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics.

The effects of phospholipid acyl chain length (n(c)), degree of acyl chain saturation, and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal acyl chain length of monounsaturated phosphatidylcholine in the absence of cholesterol was found to be 22 but decreased to 18 in the presence of 40 mol % cholesterol. This indicates that the hydrophobic matching of the lipid bilayer and the transmembrane hydrophobic core of the membrane protein is a crucial parameter in supporting optimal Na,K-ATPase activity. In addition, the increased bilayer order induced by both cholesterol and saturated phospholipids could be important for the conformational mobility of the Na,K-ATPase changing the distribution of conformations. Lipid fluidity was important for several parameters of reconstitution, e.g., the amount of protein inserted and the orientation in the liposomes. The temperature dependence of the Na,K-ATPase as well of the Na-ATPase reactions depends both on phospholipid acyl chain length and on cholesterol. Cholesterol increased significantly both the enthalpy of activation and entropy of activation for Na,K-ATPase activity and Na-ATPase activity of Na,K-ATPase reconstituted with monounsaturated phospholipids. In the presence of cholesterol the free energy of activation was minimum at a lipid acyl chain length of 18, the same that supported maximum turnover. In the case of ATPase reconstituted without cholesterol, the minimum free energy of activation and the maximum turnover both shifted to longer acyl chain lengths of about 22.

Adenosine Triphosphatases↗